Expansion of tumor infiltrating lymphocytes from liquid tumors and therapeutic uses thereof

Abstract

Methods of expanding peripheral blood lymphocytes (PBLs) from blood of patients with hematological malignancies, including lymphomas and leukemias, genetic modifications of expanded PBLs to incorporate chimeric antigen receptors, genetically modified T cell receptors, and other genetic modifications, and uses of such expanded and / or modified PBLs in the treatment of diseases such as cancers and hematological malignancies are disclosed herein.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 382,487, filed Nov. 4, 2022, all of which is herein incorporated by reference in its entirety.FIELD OF THE INVENTION

[0002] Methods of expanding peripheral blood lymphocytes (PBLs) derived from blood and / or bone marrow of a patient with a hematological malignancy, such as a liquid tumor, including lymphomas and leukemias, and compositions comprising populations of PBLs obtained therefrom, are disclosed herein. In addition, therapeutic uses of autologous PBLs expanded from blood of a patient in the treatment of hematological malignancies are disclosed herein.BACKGROUND OF THE INVENTION

[0003] Treatment of bulky, refractory cancers using adoptive autologous transfer of tumor infiltrating lymphocytes (TILs) represents a powerful approach to therapy for patients with poor prognoses. Gattinoni, et al., Nat. Rev. Immunol. 2006, 6, 383-393. TILs are dominated by T cells, and IL-2-based TIL expansion followed by a “rapid expansion process” (REP) has become a preferred method for TIL expansion because of its speed and efficiency. Dudley, et al., Science 2002, 298, 850-54; Dudley, et al., J Clin. Oncol. 2005, 23, 2346-57; Dudley, et al., J. Clin. Oncol. 2008, 26, 5233-39; Riddell, et al., Science 1992, 257, 238-41; Dudley, et al., J. Immunother. 2003, 26, 332-42. A number of approaches to improve responses to TIL therapy in melanoma and to expand TIL therapy to other tumor types have been explored with limited success, and the field remains challenging. Goff, et al., J. Clin. Oncol. 2016, 34, 2389-97; Dudley, et al., J. Clin. Oncol. 2008, 26, 5233-39; Rosenberg, et al., Clin. Cancer Res. 2011, 17, 4550-57. Earlier approaches to expansions of TILs from B cell lymphomas yielded poor results, with only 2 of 12 attempts at TIL growth providing for potential activity against tumors. Schwartzentruber, et al., Blood 1993, 82, 1204-1211. There is an urgent need to provide for more efficacious therapies in many hematological malignancies, including chronic lymphocytic leukemia (CLL). There is also an urgent need to provide such therapies using whole blood as a source of lymphocytes with TIL functionality, such as PBLs, to treat patients refractory to other therapies or that have relapsed. Because of the burden of apheresis and the large blood volumes taken from critically ill cancer patients, there is also an urgent need to use as little as patient blood as possible.

[0004] The present invention provides the surprising finding that PBLs expansion processes using low volumes of blood as a source of PBLs can result in efficacious PBL populations obtained from hematological malignancies, such as liquid tumors, including lymphomas or leukemias.SUMMARY OF THE INVENTION

[0005] In an embodiment, a method of treating a hematological malignancy in a patient is disclosed. The method comprises administering a therapeutically effective population of peripheral blood lymphocytes (PBLs) obtained from a patient pre-treated with an ITK inhibitor, a BTK inhibitor and / or a BCL2 inhibitor to the patient. The ITK inhibitor maybe an ITK inhibitor that covalently binds to ITK. The BTK inhibitor maybe a BTK inhibitor that covalently binds to BTK. The BCL2 inhibitor maybe a BCL2 inhibitor that mimics BH3.

[0006] The patient may be suffering from a leukemia or a chronic lymphocytic leukemia. The patient may have relapsed after treatment with a BTK inhibitor, a BCL2 inhibitor or a combination of a BTK inhibitor and BCL2 inhibitor. The patient may be ineligible for CAR-T therapy.

[0007] The patient may be suffering from small lymphocytic lymphoma. The patient may have relapsed after treatment with a BTK inhibitor, a BCL2 inhibitor or a combination of a BTK inhibitor and BCL2 inhibitor. The patient is ineligible for CAR-T therapy.

[0008] The patient may have been pretreated with a kinase inhibitor selected from the group consisting of imatinib, dasatinib, ibrutinib, bosutinib, nilotinib, erlotinib, acalabrutinib, zanubrutinib, tirabrutinib, orelabrutinib, evobrutinib, spebrutinib, remibrutinib, tolebrutinib, olmutinib, branebrutinib, TAK-020, elsubrutinib, rilzabrutinib, vecabrutinib, fenebrutinib, GNE-431, RN-486, BMS935177, BMS-986142, CGI-1746, GDC-0834, G-744, G-278, and a combination thereof, or a BCL2 inhibitor selected from the group consisting of venetoclax, obatoclax, subatoclax, maritoclax, gossypol, apogossypol, TW-37, UMI-77, BDA-366, navitoclax, ABT-737, and a combination hereof.

[0009] The patient may have replapsed after treatment with a kinase inhibitor selected from the group consisting of imatinib, dasatinib, ibrutinib, bosutinib, nilotinib, erlotinib, acalabrutinib, zanubrutinib, tirabrutinib, orelabrutinib, evobrutinib, spebrutinib, remibrutinib, tolebrutinib, olmutinib, branebrutinib, TAK-020, elsubrutinib, rilzabrutinib, vecabrutinib, fenebrutinib, GNE-431, RN-486, BMS935177, BMS-986142, CGI-1746, GDC-0834, G-744, G-278, and a combination thereof, or a BCL2 inhibitor selected from the group consisting of venetoclax, obatoclax, subatoclax, maritoclax, gossypol, apogossypol, TW-37, UMI-77, BDA-366, navitoclax, ABT-737, and a combinationt hereof.

[0010] In an embodiment of the invention, a method for expanding peripheral blood lymphocytes (PBLs) from peripheral blood is disclosed. In one embodiment, the method comprises (a) obtaining a sample of peripheral blood mononuclear cells (PBMCs) from the peripheral blood of a patient, wherein said sample is optionally cryopreserved and the patient is optionally pretreated with an ITK inhibitor; (b) optionally washing the PBMCs by centrifugation, (c) admixing magnetic beads selective for CD3 and CD28 to the PBMCs to form an admixture of the beads and the PBMCs; (d) seeding the admixture of the beads and the PBMCs into a gas-permeable container and co-culturing said PBMCs in media comprising about 3000 IU / mL of IL-2 in for about 4 to about 6 days; (e) feeding said PBMCs using media comprising about 3000 IU / mL of IL-2, and co-culturing said PBMCs for about 5 days, such that the total co-culture period of steps (d) and (e) is about 9 to about 11 days; (f) harvesting PBMCs from media; (g) removing the magnetic beads selective for CD3 and CD28 using a magnet; (h) removing residual B-cells using magnetic-activated cell sorting and beads selective for CD19 to provide a PBL product; (i) washing and concentrating the PBL product using a cell harvester; and (j) formulating and optionally cryopreserving the PBL product. In one embodiment, the ITK inhibitor is optionally an ITK inhibitor that covalently binds to ITK.

[0011] In one embodiment, the method comprises (a) obtaining a sample of peripheral blood mononuclear cells (PBMCs) from the peripheral blood of a patient, wherein said sample is optionally cryopreserved and the patient is optionally pretreated with an ITK inhibitor; (b) optionally washing the PBMCs by centrifugation; (c) removing B-cells from the PBMCs by selecting against CD19 to provide PBMCs depleted of B-cells; (d) admixing magnetic beads selective for CD3 and CD28 to the PBMCs depleted of B-cells to form an admixture of the beads and the PBMCs; (e) seeding the admixture of the beads and the PBMCs into a gas-permeable container and co-culturing said PBMCs in media comprising about 3000 IU / mL of IL-2 in for about 4 to about 6 days; (f) feeding said PBMCs using media comprising about 3000 IU / mL of IL-2, and co-culturing said PBMCs for about 5 days, such that the total co-culture period of steps (e) and (f) is about 9 to about 11 days; (g) harvesting the PBMCs from media; (h) removing any residual magnetic beads selective for CD3 and CD28 from the PBMCs using a magnet to provide a PBL product; (i) washing and concentrating the PBL product using a cell harvester; and (j) formulating and optionally cryopreserving the PBL product. In one embodiment, the ITK inhibitor is optionally an ITK inhibitor that covalently binds to ITK. In another embodiment, the removal of B-cells in step (c) is performed by using beads selective for CD19 to remove B-cells from the PBMCs. In another embodiment, the removal of B-cells in step (c) is performed by admixing the beads selective for CD19 with the PBMCs to form complexes of beads and B-cells in an admixture with the PBMCs and removing the complexes from the admixture. In another embodiment, the removal of B-cells in step (c) is performed by admixing magnetic beads selective for CD19 with the PBMCs to form complexes of magnetic beads and B-cells in the admixture and using a magnet to remove the complexes from the admixture. In an embodiment of the invention, the beads selective for CD19 are beads conjugated to anti-CD19 antibody.

[0012] In an embodiment of the invention, the amount of peripheral blood that is obtained from a patient in a method according to the present invention is between about 10 mL and 50 mL. In another embodiment, the amount of peripheral blood that is obtained from a patient is less than or equal to about 50 mL.

[0013] In an embodiment of the invention, the seeding density of the PBMCs in a method according to the present invention is about 2×105 / cm2 to about 1.6×103 / cm2 relative to the surface area of the gas-permeable container.

[0014] In an embodiment of the invention, a process for the preparation of peripheral blood lymphocytes (PBLs) from a whole blood sample comprises the steps of (a) obtaining peripheral blood mononuclear cells (PBMCs) from less than or equal to about 50 mL of whole blood from a patient having a liquid tumor, wherein the patient is optionally pretreated with an ITK inhibitor; (b) admixing beads selective for CD3 and CD28 with the PBMCs, wherein the beads are added at a ratio of 3 beads:1 cell, to form an admixture of the PBMCs and the beads; (c) culturing the admixture of the PBMCs and the beads at a density of about 25,000 cells per cm2 to about 50,000 cells per cm2 on a gas-permeable surface of one or more containers containing a first cell culture medium and IL-2 for a period of about 4 days, (d) adding to each container IL-2 and a second cell culture medium that is the same as or different from the first cell culture medium and culturing for a period of about 5 days to about 7 days to form an expanded population of PBLs; and (e) harvesting from each container the expanded population of PBLs.

[0015] In an embodiment of the invention, a process for the preparation of peripheral blood lymphocytes (PBLs) from a whole blood sample comprises the steps of (a) obtaining peripheral blood mononuclear cells (PBMCs) from less than or equal to about 50 mL of whole blood from a patient having a liquid tumor, wherein the patient is optionally pretreated with an ITK inhibitor; (b) removing B-cells from the PBMCs by selecting against CD19 to provide PBMCs depleted of B-cells; (c) admixing beads selective for CD3 and CD28 to the PBMCs, wherein the beads are added at a ratio of 3 beads:1 cell, to form an admixture of the PBMCs and the beads; (d) culturing the admixture of the PBMCs and the beads at a density of about 25,000 cells per cm2 to about 50,000 cells per cm2 on a gas-permeable surface of one or more containers containing a first cell culture medium and IL-2 for a period of about 4 days; (e) adding to each container IL-2 and a second cell culture medium that is the same as or different from the first cell culture medium and culturing for a period of about 5 days to about 7 days to form an expanded population of PBLs; and (f) harvesting from each container the expanded population of PBLs. In one embodiment, the ITK inhibitor is optionally an ITK inhibitor that covalently binds to ITK. In another embodiment, the patient is pretreated with an ITK inhibitor and the patient is refractory to treatment with the ITK inhibitor. In another embodiment, the removal of B-cells in step (b) is performed by using beads selective for CD19 to remove B-cells from the PBMCs. In another embodiment, the removal of B-cells in step (b) is performed by admixing the beads selective for CD19 with the PBMCs to form complexes of the beads and B-cells in an admixture with the PBMCs and removing the complexes from the admixture. In another embodiment, the removal of B-cells is performed by admixing magnetic beads selective for CD19 to the PBMCs to form complexes of the magnetic beads and B-cells in an admixture with the PBMCs and using a magnet to remove the complexes from the admixture. In another embodiment, the beads selective for CD19 are beads conjugated to anti-CD19 antibody.

[0016] In an embodiment of the method according to the present invention, the total number of cells harvested is from about 8 billion to about 22 billion.

[0017] In an embodiment of the method according to the present invention, the total number of cells harvested is from about 1 billion to about 8 billion.

[0018] In an embodiment of the method according to the present invention, about 95% to about 99% of the cells harvested are T-cells.

[0019] In an embodiment of the method according to the present invention, the step of admixing the beads selective for CD3 and CD28 to the PBMCs to form an admixture of the beads and the PBMCs is replaced with the step of admixing the beads selective for CD3 and CD28 to the PBMCs to form complexes of the beads and the PBMCs in an admixture of the beads and the PBMCs, and the step of culturing the admixture is replaced with the step of separating the complexes of the beads and the PBMCs from the admixture and culturing the complexes of the PBMCs and the beads at a density of about 25,000 cells per cm2 to about 50,000 cells per cm2 on a gas-permeable surface in one or more containers containing a first cell culture medium and IL-2 for a period of about 4 days. In another embodiment of the present invention, the beads selective for CD3 and CD28 are magnetic beads, and the step of separating the complexes of the beads and the PBMCs from the admixture is performed by using a magnet to remove the complexes from the admixture.

[0020] In an embodiment of the invention, the beads selective for CD3 and CD28 are beads conjugated to anti-CD3 antibodies and anti-CD28 antibodies.

[0021] In an embodiment of the method according to the present invention, the method further comprises performing a selection to remove any remnant B-cells from the expanded population of PBLs. In another embodiment, the selection is performed by using beads selective for CD19 to remove the remnant B-cells. In another embodiment, the selection is performed by admixing the beads selective for CD19 with the expanded population of PBLs to form complexes of beads and any remnant B-cells and removing the complexes from the admixture. In another embodiment, the selection is performed by admixing magnetic beads selective for CD19 with the expanded population of PBLs to form complexes of magnetic beads and any remnant B-cells and using a magnet to remove the complexes from the admixture. In an embodiment of the invention, the beads selective for CD19 are beads conjugated to anti-CD19 antibody.

[0022] In an embodiment according to the present invention, the first cell culture medium contains about 3000 IU / mL of IL-2. In another embodiment, the second cell culture medium contains about 3000 IU / mL of IL-2. In yet another embodiment, the cultures in the culturing steps are incubated at 37° C. and under an atmosphere containing 5% CO2.

[0023] In an embodiment of the invention, the method according to the present invention is performed over a period of about 9 to about 11 days. In another embodiment, the method is performed over a period of about 9 days. In another embodiment, the method is performed over a period of about 11 days.

[0024] In an embodiment of the invention, the patient is pretreated with an ITK inhibitor. In one embodiment, the ITK inhibitor is ibrutinib. In another embodiment, the patient has a liquid tumor. In another embodiment, the patient has a liquid tumor and is pretreated with an ITK inhibitor. In another embodiment, the patient has a liquid tumor, is refractory to treatment with an ITK inhibitor, and is pretreated with the ITK inhibitor.

[0025] In an embodiment of the invention, the patient suffers from leukemia. In another embodiment, the leukemia is chronic lymphocytic leukemia.BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings.

[0027] FIG. 1A-FIG. 1B illustrates an exemplary embodiment of a PBL manufacturing process with B-cell depletion on Day 9.

[0028] FIG. 2A-FIG. 2B illustrates the design of experiments to compare the T-cell positive selection method using CTS Dynabeads CD3 / 28 to the T-cell negative selection method using either research grade Pan-T kit or a sequential anti-CD14, anti CD19 depletion method using CliniMACS microbeads.

[0029] FIG. 3 illustrates total PBL yields (in billions) for two samples extrapolated for an exemplary 9 day manufacturing process.

[0030] FIG. 4 illustrates total PBL yields (in billions) for two samples extrapolated for an exemplary 11 day manufacturing process.

[0031] FIG. 5 illustrates total viable cell counts (TVC) of PBLs from 50 mL of whole blood from two different patients on day 9 of an exemplary manufacturing process.

[0032] FIG. 6 illustrates fold expansion of PBLs from 50 mL of whole blood from the same patients as in FIG. 5 on day 9 of an exemplary manufacturing process.

[0033] FIG. 7 illustrates interferon-gamma levels (in pg / mL / 5e5 cells) from the same patients as in FIG. 5 from an exemplary manufacturing process.

[0034] FIG. 8 illustrates interferon-gamma levels (in pg / mL) from the same patients as in FIG. 5 from an exemplary manufacturing process.

[0035] FIG. 9 illustrates an embodiment of projected doses of a PBL product.

[0036] FIG. 10A illustrates an exemplary embodiment of a PBL manufacturing process.

[0037] FIG. 10B illustrates an exemplary embodiment of a PBL manufacturing process with B-cell depletion.

[0038] FIG. 10C illustrates an exemplary embodiment of a PBL manufacturing process with B-cell depletion on Day 0.

[0039] FIG. 11 illustrates an exemplary embodiment of a PBL manufacturing process. Cryopreserved PBMCs obtained from peripheral blood of CLL patients were enriched for T-cells. Enriched fractions were expanded for a duration of 9-14 days in the presence of CTS™Dynabeads™ (αCD3 / αCD28) and IL-2 to obtain PBLs.

[0040] FIG. 12 illustrates fold expansion of PBLs using a 9 day and 14 day expansion processes in treatment-naïve, pre-ibrutinib, and post-ibrutinib treated patients. Statistical significance is shown as: *p<0.05; **p<0.01; and ***p<0.001.

[0041] FIG. 13 illustrates interferon-gamma secretion by different groups of PBLs in response to non-specific TCR engagement. IFNγ secretion was assessed using an ELIspot assay. Data shown is IFNγ secreting T-cells per million PBLs. Statistical significance is shown as: *p<0.05; **p<0.01; and ***p<0.001.

[0042] FIGS. 14A-FIG. 14H illustrate cytotoxicity of different groups of PBLs against autologous CD19+ cells. Cytotoxicity was assessed using a flow cytometry based cell-killing assay. Data samples are paired and depicted for four patients; FIGS. 14A, 14C, 14E, and 14G are pre-ibrutinib samples and FIGS. 14B, 14D, 14F, and 14H are post-ibrutinib samples.

[0043] FIG. 15 illustrates CD19+ target specificity as determined by HLA blockade experiments. HLA class I and class II molecules on cells were blocked using HLA blocking antibody cocktails.

[0044] FIGS. 16A-FIG. 16E illustrate box plots representing gene expression levels related to different T-cell pathways as measured by nCounter CAR-T characterization panel. Gene expression is represented by the y-axis score. Scores were measured for melanoma TIL (labeled as “Final”), 14-day expanded PBLs from ibrutinib-treated patients (labeled as D.14), and 9-day expanded PBLs from ibrutinib-treated patients (labeled as D.9).

[0045] FIG. 17 illustrates fold expansion in a 9 day expansion process with B-cell depletion at Day 0. Circles represent IRuns 1,2, 4, and 5, and the square represents MRun 5. As shown, B-cell depletion at Day 0, particularly in samples with a high initial B-cell content, does not appear to negatively affect fold expansion of T-cells over the 9 day process, even though the initial T-cell content may be partially depleted in the Day 0 B-cell depletion process (see FIG. 18).

[0046] FIGS. 18A and 18B illustrate Day 9 T cell yields versus total initial T-cells (FIG. 18A) and initial B-cell content (FIG. 18B) with B-cell depletion occurring either at Day 0 or at Day 9.BRIEF DESCRIPTION OF THE SEQUENCE LISTINGSEQ ID NO:1 is the amino acid sequence of the heavy chain of muromonab.

[0048] SEQ ID NO:2 is the amino acid sequence of the light chain of muromonab.

[0049] SEQ ID NO:3 is the amino acid sequence of a recombinant human IL-2 protein.

[0050] SEQ ID NO:4 is the amino acid sequence of aldesleukin.

[0051] SEQ ID NO:5 is the amino acid sequence of a recombinant human IL-4 protein.

[0052] SEQ ID NO:6 is the amino acid sequence of a recombinant human IL-7 protein.

[0053] SEQ ID NO:7 is the amino acid sequence of a recombinant human IL-15 protein.

[0054] SEQ ID NO:8 is the amino acid sequence of a recombinant human IL-21 protein.DETAILED DESCRIPTION OF THE INVENTION

[0055] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs. All patents and publications referred to herein are incorporated by reference in their entireties.Definitions

[0056] The terms “co-administration,”“co-administering,”“administered in combination with,”“administering in combination with,”“simultaneous,” and “concurrent,” as used herein, encompass administration of two or more active pharmaceutical ingredients to a subject so that both active pharmaceutical ingredients and / or their metabolites are present in the subject at the same time. Co-administration includes simultaneous administration in separate compositions, administration at different times in separate compositions, or administration in a composition in which two or more active pharmaceutical ingredients are present. Simultaneous administration in separate compositions and administration in a composition in which both agents are present are preferred.

[0057] The term “in vivo” refers to an event that takes place in a mammalian subject's body.

[0058] The term “ex vivo” refers to an event that takes place outside of a mammalian subject's body, in an artificial environment.

[0059] The term “in vitro” refers to an event that takes places in a test system. In vitro assays encompass cell-based assays in which alive or dead cells may be are employed and may also encompass a cell-free assay in which no intact cells are employed.

[0060] The term “rapid expansion” means an increase in the number of antigen-specific TILs of at least about 3-fold (or 4-, 5-, 6-, 7-, 8-, or 9-fold) over a period of a week, more preferably at least about 10-fold (or 20-, 30-, 40-, 50-, 60-, 70-, 80-, or 90-fold) over a period of a week, or most preferably at least about 100-fold over a period of a week. A number of rapid expansion protocols are described herein.

[0061] The terms “fragmenting,”“fragment,” and “fragmented,” as used herein to describe processes for disrupting a tumor, includes mechanical fragmentation methods such as crushing, slicing, dividing, and morcellating tumor tissue as well as any other method for disrupting the physical structure of tumor tissue.

[0062] The terms “peripheral blood mononuclear cells” and “PBMCs” refers to a peripheral blood cell having a round nucleus, including lymphocytes (T cells, B cells, NK cells) and monocytes. Optionally, the peripheral blood mononuclear cells are irradiated allogeneic peripheral blood mononuclear cells. PBMCs include antigen presenting cells. The term “PBLs” refers to peripheral blood lymphocytes and are T-cells expanded from peripheral blood. The terms PBL and TIL are used interchangeably herein.

[0063] The term “anti-CD3 antibody” refers to an antibody or variant thereof, e.g., a monoclonal antibody and including human, humanized, chimeric or murine antibodies which are directed against the CD3 receptor in the T cell antigen receptor of mature T cells. Anti-CD3 antibodies include OKT-3, also known as muromonab, and UCHT-1. Other anti-CD3 antibodies include, for example, otelixizumab, teplizumab, and visilizumab.

[0064] The term “OKT-3” (also referred to herein as “OKT3”) refers to a monoclonal antibody or biosimilar or variant thereof, including human, humanized, chimeric, or murine antibodies, directed against the CD3 receptor in the T cell antigen receptor of mature T cells, and includes commercially-available forms such as OKT-3 (30 ng / mL, MACS GMP CD3 pure, Miltenyi Biotech, Inc., San Diego, CA, USA) and muromonab or variants, conservative amino acid substitutions, glycoforms, or biosimilars thereof. The amino acid sequences of the heavy and light chains of muromonab are given in Table 1 (SEQ ID NO:1 and SEQ ID NO:2). A hybridoma capable of producing OKT-3 is deposited with the American Type Culture Collection and assigned the ATCC accession number CRL 8001. A hybridoma capable of producing OKT-3 is also deposited with European Collection of Authenticated Cell Cultures (ECACC) and assigned Catalogue No. 86022706.TABLE 1Amino acid sequences of muromonab.IdentifierSequence (One-Letter Amino Acid Symbols)SEQ ID NO: 1QVQLQQSGAE LARPGASVKM SCKASGYTFT RYTMHWVKQR PGQGLEWIGY INPSRGYTNY  60Muromonab NQKFKDKATL TTDKSSSTAY MQLSSLTSED SAVYYCARYY DDHYCLDYWG QGTTLTVSSA 120heavy chainKTTAPSVYPL APVCGGTTGS SVTLGCLVKG YFPEPVTLTW NSGSLSSGVH TFPAVLQSDL 180YTLSSSVTVT SSTWPSQSIT CNVAHPASST KVDKKIEPRP KSCDKTHTCP PCPAPELLGG 240PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN 300STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE 360LTXNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW 420QQGNVFSCSV MHEALHNHYT QKSLSLSPGK                                  450SEQ ID NO: 2QIVLTQSPAI MSASPGEKVT MTCSASSSVS YMNWYQQKSG TSPKRWIYDT SKLASGVPAH  60Muromonab FRGSGSGTSY SLTISGMEAE DAATYYCQQW SSNPFTFGSG TKLEINRADT APTVSIFPPS 120light chainSEQLTSGGAS VVCFLNNFYP KDINVKWKID GSERQNGVLN SWTDQDSKDS TYSMSSTLTL 180TKDEYERHNS YTCEATHKTS TSPIVKSENR NEC                              213

[0065] The term “IL-2” (also referred to herein as “IL2”) refers to the T cell growth factor known as interleukin-2, and includes all forms of IL-2 including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants thereof. IL-2 is described, e.g., in Nelson, J. Immunol. 2004, 172, 3983-88 and Malek, Annu. Rev. Immunol. 2008, 26, 453-79, the disclosures of which are incorporated by reference herein. The amino acid sequence of recombinant human IL-2 suitable for use in the invention is given in Table 2 (SEQ ID NO:3). For example, the term IL-2 encompasses human, recombinant forms of IL-2 such as aldesleukin (PROLEUKIN, available commercially from multiple suppliers in 22 million JU per single use vials), as well as the form of recombinant IL-2 commercially supplied by CellGenix, Inc., Portsmouth, NH, USA (CELLGRO GMP) or ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (Cat. No. CYT-209-b) and other commercial equivalents from other vendors. Aldesleukin (des-alanyl-1, serine-125 human IL-2) is a nonglycosylated human recombinant form of IL-2 with a molecular weight of approximately 15 kDa. The amino acid sequence of aldesleukin suitable for use in the invention is given in Table 2 (SEQ ID NO:4). The term IL-2 also encompasses pegylated forms of IL-2, as described herein, including the pegylated IL-2 prodrug NKTR-214, available from Nektar Therapeutics, South San Francisco, CA, USA. NKTR-214 and pegylated IL-2 suitable for use in the invention is described in U.S. Patent Application Publication No. US 2014 / 0328791 A1 and International Patent Application Publication No. WO 2012 / 065086 A1, the disclosures of which are incorporated by reference herein. Alternative forms of conjugated IL-2 suitable for use in the invention are described in U.S. Pat. Nos. 4,766,106, 5,206,344, 5,089,261 and 4,902,502, the disclosures of which are incorporated by reference herein. Formulations of IL-2 suitable for use in the invention are described in U.S. Pat. No. 6,706,289, the disclosure of which is incorporated by reference herein.TABLE 2Amino acid sequences of interleukins.IdentifierSequence (One-Letter Amino Acid Symbols)SEQ ID NO: 3MAPTSSSTKK TQLQLEHLLL DLQMILNGIN NYKNPKLTRM LTFKFYMPKK ATELKHLQCL  60recombinantEEELKPLEEV LNLAQSKNFH LRPRDLISNI NVIVLELKGS ETTFMCEYAD ETATIVEFLN 120human IL-2RWITFCQSII STLT                                                   134(rhIL-2)SEQ ID NO: 4PTSSSTKKTQ LQLEHLLLDL QMILNGINNY KNPKLTRMLT FKFYMPKKAT ELKHLQCLEE  60AldesleukinELXPLEEVLN LAQSKNFHLR PRDLISNINV IVLELKGSET TFMCEYADET ATIVEFLNRW 120ITFSQSIIST LT                                                     132SEQ ID NO: 5MHKCDITLQE IIKTLNSLTE QKTLCTELTV TDIFAASKNT TEKETFCRAA TVLRQFYSHH  60human IL-4EKDTRCLGAT AQQFHRHKQL IRFLKRLDRN LWGLAGLNSC PVKEANQSTL ENFLERLKTI 120recombinantMREKYSKCSS                                                        130human IL-4(rhIL-4)SEQ ID NO: 6MDCDIEGKDG KQYESVLMVS IDQLLDSMKE IGSNCLNNEF NFFKRHICDA NKEGMFLFRA  60recombinantARKLRQFLKM NSTGDFDLHL LKVSEGTTIL LNCTGQVKGR KPAALGEAQP TKSLEENKSL 120human IL-7KEQKKLNDLC FLKRLLQEIK TQWNKILMGT KEH                              153(rhIL-7)SEQ ID NO: 7MNWVNVISDL KKIEDLIQSM HIDATLYTES DVHPSCKVTA MKCFLLELQV ISLESGDASI  60recombinantHDTVENLIIL ANNSLSSNGN VTESGCKECE ELEEKNIKEF LQSFVHIVQM FINTS      115human IL-15(rhIL-15)SEQ ID NO: 8MQDRHMIRMR QLIDIVDQLK NYVNDLVPEF LPAPEDVETN CEWSAFSCFQ KAQLKSANTG  60recombinantNNERIINVSI KKLKRKPPST NAGRRQKHRL TCPSCDSYEK KPPKEFLERF KSLLQKMIHQ 120human IL-21HLSSRTHGSE DS                                                     132(rhIL-21)

[0066] The term “IL-4” (also referred to herein as “IL4”) refers to the cytokine known as interleukin 4, which is produced by Th2 T cells and by eosinophils, basophils, and mast cells. IL-4 regulates the differentiation of naïve helper T cells (Th0 cells) to Th2 T cells. Steinke and Borish, Respir. Res. 2001, 2, 66-70. Upon activation by IL-4, Th2 T cells subsequently produce additional IL-4 in a positive feedback loop. IL-4 also stimulates B cell proliferation and class II MHC expression, and induces class switching to IgE and IgG1 expression from B cells. Recombinant human IL-4 suitable for use in the invention is commercially available from multiple suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (Cat. No. CYT-211) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-15 recombinant protein, Cat. No. Gibco CTP0043). The amino acid sequence of recombinant human IL-4 suitable for use in the invention is given in Table 2 (SEQ ID NO:5).

[0067] The term “IL-7” (also referred to herein as “IL7”) refers to a glycosylated tissue-derived cytokine known as interleukin 7, which may be obtained from stromal and epithelial cells, as well as from dendritic cells. Fry and Mackall, Blood 2002, 99, 3892-904. IL-7 can stimulate the development of T cells. IL-7 binds to the IL-7 receptor, a heterodimer consisting of IL-7 receptor alpha and common gamma chain receptor, which in a series of signals important for T cell development within the thymus and survival within the periphery. Recombinant human IL-7 suitable for use in the invention is commercially available from multiple suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (Cat. No. CYT-254) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-7 recombinant protein, Cat. No. Gibco PHC0071). The amino acid sequence of recombinant human IL-7 suitable for use in the invention is given in Table 2 (SEQ ID NO:6).

[0068] The term “IL-15” (also referred to herein as “IL15”) refers to the T cell growth factor known as interleukin-15, and includes all forms of IL-15 including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants thereof. IL-15 is described, e.g., in Fehniger and Caligiuri, Blood 2001, 97, 14-32, the disclosure of which is incorporated by reference herein. IL-15 shares P and 7 signaling receptor subunits with IL-2. Recombinant human IL-15 is a single, non-glycosylated polypeptide chain containing 114 amino acids (and an N-terminal methionine) with a molecular mass of 12.8 kDa. Recombinant human IL-15 is commercially available from multiple suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (Cat. No. CYT-230-b) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-15 recombinant protein, Cat. No. 34-8159-82). The amino acid sequence of recombinant human IL-15 suitable for use in the invention is given in Table 2 (SEQ ID NO:7).

[0069] The term “IL-21” (also referred to herein as “IL21”) refers to the pleiotropic cytokine protein known as interleukin-21, and includes all forms of IL-21 including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants thereof. IL-21 is described, e.g., in Spolski and Leonard, Nat. Rev. Drug. Disc. 2014, 13, 379-95, the disclosure of which is incorporated by reference herein. IL-21 is primarily produced by natural killer T cells and activated human CD4+ T cells. Recombinant human IL-21 is a single, non-glycosylated polypeptide chain containing 132 amino acids with a molecular mass of 15.4 kDa. Recombinant human IL-21 is commercially available from multiple suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (Cat. No. CYT-408-b) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-21 recombinant protein, Cat. No. 14-8219-80). The amino acid sequence of recombinant human IL-21 suitable for use in the invention is given in Table 2 (SEQ ID NO:8).

[0070] The terms “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” are intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and inert ingredients. The use of such pharmaceutically acceptable carriers or pharmaceutically acceptable excipients for active pharmaceutical ingredients is well known in the art. Except insofar as any conventional pharmaceutically acceptable carrier or pharmaceutically acceptable excipient is incompatible with the active pharmaceutical ingredient, its use in the therapeutic compositions of the invention is contemplated. Additional active pharmaceutical ingredients, such as other drugs, can also be incorporated into the described compositions and methods.

[0071] The terms “antibody” and its plural form “antibodies” refer to whole immunoglobulins and any antigen-binding fragment (“antigen-binding portion”) or single chains thereof. An “antibody” further refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions of an antibody may be further subdivided into regions of hypervariability, which are referred to as complementarity determining regions (CDR) or hypervariable regions (HVR), and which can be interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen epitope or epitopes. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0072] The term “antigen” refers to a substance that induces an immune response. In some embodiments, an antigen is a molecule capable of being bound by an antibody or a TCR if presented by major histocompatibility complex (MHC) molecules. The term “antigen”, as used herein, also encompasses T cell epitopes. An antigen is additionally capable of being recognized by the immune system. In some embodiments, an antigen is capable of inducing a humoral immune response or a cellular immune response leading to the activation of B lymphocytes and / or T lymphocytes. In some cases, this may require that the antigen contains or is linked to a Th cell epitope. An antigen can also have one or more epitopes (e.g., B- and T-epitopes). In some embodiments, an antigen will preferably react, typically in a highly specific and selective manner, with its corresponding antibody or TCR and not with the multitude of other antibodies or TCRs which may be induced by other antigens.

[0073] The terms “monoclonal antibody,”“mAb,”“monoclonal antibody composition,” or their plural forms refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope. Monoclonal antibodies specific to certain receptors can be made using knowledge and skill in the art of injecting test subjects with suitable antigen and then isolating hybridomas expressing antibodies having the desired sequence or functional characteristics. DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the monoclonal antibodies). The hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. Recombinant production of antibodies will be described in more detail below.

[0074] The terms “antigen-binding portion” or “antigen-binding fragment” of an antibody (or simply “antibody portion” or “fragment”), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term “antigen-binding portion” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a domain antibody (dAb) fragment (Ward, et al., Nature, 1989, 341, 544-546), which may consist of a VH or a VL domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules known as single chain Fv (scFv); see, e.g., Bird, et al., Science 1988, 242, 423-426; and Huston, et al., Proc. Natl. Acad. Sci. USA 1988, 85, 5879-5883). Such scFv antibodies are also intended to be encompassed within the terms “antigen-binding portion” or “antigen-binding fragment” of an antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[0075] The term “human antibody,” as used herein, is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). The term “human antibody”, as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0076] The term “human monoclonal antibody” refers to antibodies displaying a single binding specificity which have variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. In an embodiment, the human monoclonal antibodies are produced by a hybridoma which includes a B cell obtained from a transgenic nonhuman animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell.

[0077] The term “recombinant human antibody”, as used herein, includes all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an animal (such as a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom (described further below), (b) antibodies isolated from a host cell transformed to express the human antibody, e.g., from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial human antibody library, and (d) antibodies prepared, expressed, created or isolated by any other means that involve splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.

[0078] As used herein, “isotype” refers to the antibody class (e.g., IgM or IgG1) that is encoded by the heavy chain constant region genes.

[0079] The phrases “an antibody recognizing an antigen” and “an antibody specific for an antigen” are used interchangeably herein with the term “an antibody which binds specifically to an antigen.”

[0080] The term “human antibody derivatives” refers to any modified form of the human antibody, including a conjugate of the antibody and another active pharmaceutical ingredient or antibody. The terms “conjugate,”“antibody-drug conjugate”, “ADC,” or “immunoconjugate” refers to an antibody, or a fragment thereof, conjugated to another therapeutic moiety, which can be conjugated to antibodies described herein using methods available in the art.

[0081] The terms “humanized antibody,”“humanized antibodies,” and “humanized” are intended to refer to antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Additional framework region modifications may be made within the human framework sequences. Humanized forms of non-human (for example, murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a 15 hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones, et al., Nature 1986, 321, 522-525; Riechmann, et al., Nature 1988, 332, 323-329; and Presta, Curr. Op. Struct. Biol. 1992, 2, 593-596. The antibodies described herein may also be modified to employ any Fc variant which is known to impart an improvement (e.g., reduction) in effector function and / or FcR binding. The Fc variants may include, for example, any one of the amino acid substitutions disclosed in International Patent Application Publication Nos. WO 1988 / 07089 A1, WO 1996 / 14339 A1, WO 1998 / 05787 A1, WO 1998 / 23289 A1, WO 1999 / 51642 A1, WO 99 / 58572 A1, WO 2000 / 09560 A2, WO 2000 / 32767 A1, WO 2000 / 42072 A2, WO 2002 / 44215 A2, WO 2002 / 060919 A2, WO 2003 / 074569 A2, WO 2004 / 016750 A2, WO 2004 / 029207 A2, WO 2004 / 035752 A2, WO 2004 / 063351 A2, WO 2004 / 074455 A2, WO 2004 / 099249 A2, WO 2005 / 040217 A2, WO 2005 / 070963 A1, WO 2005 / 077981 A2, WO 2005 / 092925 A2, WO 2005 / 123780 A2, WO 2006 / 019447 A1, WO 2006 / 047350 A2, and WO 2006 / 085967 A2; and U.S. Pat. Nos. 5,648,260; 5,739,277; 5,834,250; 5,869,046; 6,096,871; 6,121,022; 6,194,551; 6,242,195; 6,277,375; 6,528,624; 6,538,124; 6,737,056; 6,821,505; 6,998,253; and 7,083,784; the disclosures of which are incorporated by reference herein.

[0082] The term “chimeric antibody” is intended to refer to antibodies in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as an antibody in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody.

[0083] A “diabody” is a small antibody fragment with two antigen-binding sites. The fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL or VL-VH). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described more fully in, e.g., European Patent No. EP 404,097, International Patent Publication No. WO 93 / 11161; and Bolliger, et al., Proc. Natl. Acad. Sci. USA 1993, 90, 6444-6448.

[0084] The term “glycosylation” refers to a modified derivative of an antibody. An aglycoslated antibody lacks glycosylation. Glycosylation can be altered to, for example, increase the affinity of the antibody for antigen. Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site. Aglycosylation may increase the affinity of the antibody for antigen, as described in U.S. Pat. Nos. 5,714,350 and 6,350,861. Additionally or alternatively, an antibody can be made that has an altered type of glycosylation, such as a hypofucosylated antibody having reduced amounts of fucosyl residues or an antibody having increased bisecting GlcNac structures. Such altered glycosylation patterns have been demonstrated to increase the ability of antibodies. Such carbohydrate modifications can be accomplished by, for example, expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which to express recombinant antibodies of the invention to thereby produce an antibody with altered glycosylation. For example, the cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene, FUT8 (alpha (1,6) fucosyltransferase), such that antibodies expressed in the Ms704, Ms705, and Ms709 cell lines lack fucose on their carbohydrates. The Ms704, Ms705, and Ms709 FUT8− / − cell lines were created by the targeted disruption of the FUT8 gene in CHO / DG44 cells using two replacement vectors (see e.g. U.S. Patent Publication No. 2004 / 0110704 or Yamane-Ohnuki, et al., Biotechnol. Bioeng., 2004, 87, 614-622). As another example, European Patent No. EP 1,176,195 describes a cell line with a functionally disrupted FUT8 gene, which encodes a fucosyl transferase, such that antibodies expressed in such a cell line exhibit hypofucosylation by reducing or eliminating the alpha 1,6 bond-related enzyme, and also describes cell lines which have a low enzyme activity for adding fucose to the N-acetylglucosamine that binds to the Fc region of the antibody or does not have the enzyme activity, for example the rat myeloma cell line YB2 / 0 (ATCC CRL 1662). International Patent Publication WO 03 / 035835 describes a variant CHO cell line, Lec 13 cells, with reduced ability to attach fucose to Asn(297)-linked carbohydrates, also resulting in hypofucosylation of antibodies expressed in that host cell (see also Shields, et al., J. Biol. Chem. 2002, 277, 26733-26740. International Patent Publication WO 99 / 54342 describes cell lines engineered to express glycoprotein-modifying glycosyl transferases (e.g., beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) such that antibodies expressed in the engineered cell lines exhibit increased bisecting GlcNac structures which results in increased ADCC activity of the antibodies (see also Umana, et al., Nat. Biotech. 1999, 17, 176-180). Alternatively, the fucose residues of the antibody may be cleaved off using a fucosidase enzyme. For example, the fucosidase alpha-L-fucosidase removes fucosyl residues from antibodies as described in Tarentino, et al., Biochem. 1975, 14, 5516-5523.

[0085] “Pegylation” refers to a modified antibody, or a fragment thereof, that typically is reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the antibody or antibody fragment. Pegylation may, for example, increase the biological (e.g., serum) half life of the antibody. Preferably, the pegylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term “polyethylene glycol” is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (C1-C10)alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. The antibody to be pegylated may be an aglycosylated antibody. Methods for pegylation are known in the art and can be applied to the antibodies of the invention, as described for example in European Patent Nos. EP 0154316 and EP 0401384 and U.S. Pat. No. 5,824,778, the disclosures of each of which are incorporated by reference herein.

[0086] The terms “fusion protein” or “fusion polypeptide” refer to proteins that combine the properties of two or more individual proteins. Such proteins have at least two heterologous polypeptides covalently linked either directly or via an amino acid linker. The polypeptides forming the fusion protein are typically linked C-terminus to N-terminus, although they can also be linked C-terminus to C-terminus, N-terminus to N-terminus, or N-terminus to C-terminus. The polypeptides of the fusion protein can be in any order and may include more than one of either or both of the constituent polypeptides. The term encompasses conservatively modified variants, polymorphic variants, alleles, mutants, subsequences, interspecies homologs, and immunogenic fragments of the antigens that make up the fusion protein. Fusion proteins of the disclosure can also comprise additional copies of a component antigen or immunogenic fragment thereof. The fusion protein may contain one or more binding domains linked together and further linked to an Fc domain, such as an IgG Fc domain. Fusion proteins may be further linked together to mimic a monoclonal antibody and provide six or more binding domains. Fusion proteins may be produced by recombinant methods as is known in the art. Preparation of fusion proteins are known in the art and are described, e.g., in International Patent Application Publication Nos. WO 1995 / 027735 A1, WO 2005 / 103077 A1, WO 2008 / 025516 A1, WO 2009 / 007120 A1, WO 2010 / 003766 A1, WO 2010 / 010051 A1, WO 2010 / 078966 A1, U.S. Patent Application Publication Nos. US 2015 / 0125419 A1 and US 2016 / 0272695 A1, and U.S. Pat. No. 8,921,519, the disclosures of each of which are incorporated by reference herein.

[0087] The term “heterologous” when used with reference to portions of a nucleic acid or protein indicates that the nucleic acid or protein comprises two or more subsequences that are not found in the same relationship to each other in nature. For instance, the nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source, or coding regions from different sources. Similarly, a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).

[0088] The term “conservative amino acid substitutions” in means amino acid sequence modifications which do not abrogate the binding of an antibody or fusion protein to the antigen. Conservative amino acid substitutions include the substitution of an amino acid in one class by an amino acid of the same class, where a class is defined by common physicochemical amino acid side chain properties and high substitution frequencies in homologous proteins found in nature, as determined, for example, by a standard Dayhoff frequency exchange matrix or BLOSUM matrix. Six general classes of amino acid side chains have been categorized and include: Class I (Cys); Class II (Ser, Thr, Pro, Ala, Gly); Class III (Asn, Asp, Gln, Glu); Class IV (His, Arg, Lys); Class V (Ile, Leu, Val, Met); and Class VI (Phe, Tyr, Trp). For example, substitution of an Asp for another class III residue such as Asn, Gln, or Glu, is a conservative substitution. Thus, a predicted nonessential amino acid residue in an antibody is preferably replaced with another amino acid residue from the same class. Methods of identifying amino acid conservative substitutions which do not eliminate antigen binding are well-known in the art (see, e.g., Brummell, et al., Biochemistry 1993, 32, 1180-1187; Kobayashi, et al., Protein Eng. 1999, 12, 879-884 (1999); and Burks, et al., Proc. Natl. Acad. Sci. USA 1997, 94, 412-417.

[0089] The terms “sequence identity,”“percent identity,” and “sequence percent identity” (or synonyms thereof, e.g., “99% identical”) in the context of two or more nucleic acids or polypeptides, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned (introducing gaps, if necessary) for maximum correspondence, not considering any conservative amino acid substitutions as part of the sequence identity. The percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software are known in the art that can be used to obtain alignments of amino acid or nucleotide sequences. Suitable programs to determine percent sequence identity include for example the BLAST suite of programs available from the U.S. Government's National Center for Biotechnology Information BLAST web site. Comparisons between two sequences can be carried using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. ALIGN, ALIGN-2 (Genentech, South San Francisco, California) or MegAlign, available from DNASTAR, are additional publicly available software programs that can be used to align sequences. One skilled in the art can determine appropriate parameters for maximal alignment by particular alignment software. In certain embodiments, the default parameters of the alignment software are used.

[0090] As used herein, the term “variant” encompasses but is not limited to antibodies or fusion proteins which comprise an amino acid sequence which differs from the amino acid sequence of a reference antibody by way of one or more substitutions, deletions and / or additions at certain positions within or adjacent to the amino acid sequence of the reference antibody. The variant may comprise one or more conservative substitutions in its amino acid sequence as compared to the amino acid sequence of a reference antibody. Conservative substitutions may involve, e.g., the substitution of similarly charged or uncharged amino acids. The variant retains the ability to specifically bind to the antigen of the reference antibody. The term variant also includes pegylated antibodies or proteins.

[0091] Nucleic acid sequences implicitly encompass conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues. Batzer, et al., Nucleic Acid Res. 1991, 19, 5081; Ohtsuka, et al., J. Biol. Chem. 1985, 260, 2605-2608; Rossolini, et al., Mol. Cell. Probes 1994, 8, 91-98. The term nucleic acid is used interchangeably with cDNA, mRNA, oligonucleotide, and polynucleotide.

[0092] The term “biosimilar” means a biological product, including a monoclonal antibody or protein, that is highly similar to a U.S. licensed reference biological product notwithstanding minor differences in clinically inactive components, and for which there are no clinically meaningful differences between the biological product and the reference product in terms of the safety, purity, and potency of the product. Furthermore, a similar biological or “biosimilar” medicine is a biological medicine that is similar to another biological medicine that has already been authorized for use by the European Medicines Agency. The term “biosimilar” is also used synonymously by other national and regional regulatory agencies. Biological products or biological medicines are medicines that are made by or derived from a biological source, such as a bacterium or yeast. They can consist of relatively small molecules such as human insulin or erythropoietin, or complex molecules such as monoclonal antibodies. For example, if the reference IL-2 protein is aldesleukin (PROLEUKIN), a protein approved by drug regulatory authorities with reference to aldesleukin is a “biosimilar to” aldesleukin or is a “biosimilar thereof” of aldesleukin. In Europe, a similar biological or “biosimilar” medicine is a biological medicine that is similar to another biological medicine that has already been authorized for use by the European Medicines Agency (EMA). The relevant legal basis for similar biological applications in Europe is Article 6 of Regulation (EC) No 726 / 2004 and Article 10(4) of Directive 2001 / 83 / EC, as amended and therefore in Europe, the biosimilar may be authorized, approved for authorization or subject of an application for authorization under Article 6 of Regulation (EC) No 726 / 2004 and Article 10(4) of Directive 2001 / 83 / EC. The already authorized original biological medicinal product may be referred to as a “reference medicinal product” in Europe. Some of the requirements for a product to be considered a biosimilar are outlined in the CHMP Guideline on Similar Biological Medicinal Products. In addition, product specific guidelines, including guidelines relating to monoclonal antibody biosimilars, are provided on a product-by-product basis by the EMA and published on its website. A biosimilar as described herein may be similar to the reference medicinal product by way of quality characteristics, biological activity, mechanism of action, safety profiles and / or efficacy. In addition, the biosimilar may be used or be intended for use to treat the same conditions as the reference medicinal product. Thus, a biosimilar as described herein may be deemed to have similar or highly similar quality characteristics to a reference medicinal product. Alternatively, or in addition, a biosimilar as described herein may be deemed to have similar or highly similar biological activity to a reference medicinal product. Alternatively, or in addition, a biosimilar as described herein may be deemed to have a similar or highly similar safety profile to a reference medicinal product. Alternatively, or in addition, a biosimilar as described herein may be deemed to have similar or highly similar efficacy to a reference medicinal product. As described herein, a biosimilar in Europe is compared to a reference medicinal product which has been authorized by the EMA. However, in some instances, the biosimilar may be compared to a biological medicinal product which has been authorized outside the European Economic Area (a non-EEA authorized “comparator”) in certain studies. Such studies include for example certain clinical and in vivo non-clinical studies. As used herein, the term “biosimilar” also relates to a biological medicinal product which has been or may be compared to a non-EEA authorized comparator. Certain biosimilars are proteins such as antibodies, antibody fragments (for example, antigen binding portions) and fusion proteins. A protein biosimilar may have an amino acid sequence that has minor modifications in the amino acid structure (including for example deletions, additions, and / or substitutions of amino acids) which do not significantly affect the function of the polypeptide. The biosimilar may comprise an amino acid sequence having a sequence identity of 97% or greater to the amino acid sequence of its reference medicinal product, e.g., 97%, 98%, 99% or 100%. The biosimilar may comprise one or more post-translational modifications, for example, although not limited to, glycosylation, oxidation, deamidation, and / or truncation which is / are different to the post-translational modifications of the reference medicinal product, provided that the differences do not result in a change in safety and / or efficacy of the medicinal product. The biosimilar may have an identical or different glycosylation pattern to the reference medicinal product. Particularly, although not exclusively, the biosimilar may have a different glycosylation pattern if the differences address or are intended to address safety concerns associated with the reference medicinal product. Additionally, the biosimilar may deviate from the reference medicinal product in for example its strength, pharmaceutical form, formulation, excipients and / or presentation, providing safety and efficacy of the medicinal product is not compromised. The biosimilar may comprise differences in for example pharmacokinetic (PK) and / or pharmacodynamic (PD) profiles as compared to the reference medicinal product but is still deemed sufficiently similar to the reference medicinal product as to be authorized or considered suitable for authorization. In certain circumstances, the biosimilar exhibits different binding characteristics as compared to the reference medicinal product, wherein the different binding characteristics are considered by a Regulatory Authority such as the EMA not to be a barrier for authorization as a similar biological product. The term “biosimilar” is also used synonymously by other national and regional regulatory agencies.

[0093] The term “hematological malignancy” refers to mammalian cancers and tumors of the hematopoietic and lymphoid tissues, including but not limited to tissues of the blood, bone marrow, lymph nodes, and lymphatic system. Hematological malignancies may result in the formation of a “liquid tumor.” Hematological malignancies include, but are not limited to, acute lymphoblastic leukemia (ALL), chronic lymphocytic lymphoma (CLL), small lymphocytic lymphoma (SLL), acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), acute monocytic leukemia (AMoL), Hodgkin's lymphoma, and non-Hodgkin's lymphomas. The term “B cell hematological malignancy” refers to hematological malignancies that affect B cells.

[0094] The term “liquid tumor” refers to an abnormal mass of cells that is fluid in nature. Liquid tumor cancers include, but are not limited to, leukemias, myelomas, and lymphomas, as well as other hematological malignancies. TILs obtained from liquid tumors, including liquid tumors resident in bone marrow, may also be referred to herein as marrow infiltrating lymphocytes (MILs). TILs obtained from liquid tumors, including liquid tumors circulating in peripheral blood, may also be referred to herein as PBLs. The terms MIL, TIL, and PBL are used interchangeably herein and differ only based on the tissue type from which the cells are derived.

[0095] The term “biopsy” refers to any medical procedure used to obtain cancerous cells, including bone marrow biopsy.

[0096] The terms “acute myeloid leukemia” or “AML” refers to cancers of the myeloid blood cell lines, which are also known in the art as acute myelogenous leukemia and acute nonlymphocytic leukemia. Although AML is a liquid tumor, some manifestations of AML, including extramedullary manifestations such as chloroma, exhibit properties of a solid tumor, but are classified herein as a liquid tumor. AML may be classified based on patient history, morphologic findings, and the presence or absence of specific genetic abnormalities such as, for example, AML with myelodysplasia related changes (AML-MRC), listed in Table 19. In certain cases, AML may also be diagnosed in patients with history of myelodisplastic syndrome (MDS) or based on the presence of morphological dysplasia in certain cell lineages which are determined based on genetic abnormalities listed in Table 19.TABLE 19Genetic abnormalities that affect AML classification:AML with Recurrent GeneticAbnormalitiesAML with Myelodysplasia Related ChangesRUNX1-RUNX1T1 t(8; 21)(q22; q22)Complex karyotype (≥3 unrelatedabnormalities)CBFB-MYH11 inv(16)(p12.1q22) or−7 / del(7q), −5 / del(5q)t(16; 16)(p13.1; q22)PML-RARA t(15; 17)(q22; q12)−13 / del(13q), del(11q), del(12p) / t(12p), del(9q)MLLT3-MLL / KMT2A t(9; 11)(q22; q23)i(17q) / t(17p), idic(X)(q13)DEK-NUP214 t(6; 9)(p23; q34)t(5; 12)(q33; p12), t(5; 7)(q33; q11.2)t(5; 17)(q33; p13), t(5; 10)(q33; q21)RPN-EVI1 inv(3)(q21q26.2) ort(1; 3)(p36.3; q21.2), t(3; 5)(q25; q34)t(3; 3)(q21; q26.2)RBM15-MKL1 t(1; 22)(p13; q13)t(11; 16)(q23; p13.3) *, t(3; 21)(q26.2; q22.1) *NPM1 gene mutationt(2; 11)(p21; q23) *Mutated CEBPA

[0097] The terms “chronic lymphocytic lymphoma” or “CLL” refers to cancers of the blood and bone marrow. The terms “small lymphocytic lymphoma” or “SLL” refers to a slow growing (indolent) non-Hodgkin lymphoma that affects B cells. While CLL and SLL are classified as the same disease, the difference between SLL and CLL is where the blood cancer is located. CLL has most of the cancerous B lymphocytes in the bloodstream (like a leukaemia), whereas SLL has most of the cancerous B lymphocytes in the lymph nodes and lymphoid tissue such as the spleen and the tonsils (like a lymphoma). Some of the common therapeutic targets for treatment of CLL include inhibitors of Bruton tyrosine kinase (BTK), and apoptosis regulator B-cell leukemia / lymphoma 2 (BCL-2).

[0098] The term “microenvironment,” as used herein, may refer to the solid or hematological tumor microenvironment as a whole or to an individual subset of cells within the microenvironment. The tumor microenvironment, as used herein, refers to a complex mixture of “cells, soluble factors, signaling molecules, extracellular matrices, and mechanical cues that promote neoplastic transformation, support tumor growth and invasion, protect the tumor from host immunity, foster therapeutic resistance, and provide niches for dominant metastases to thrive,” as described in Swartz, et al., Cancer Res., 2012, 72, 2473. Although tumors express antigens that should be recognized by T cells, tumor clearance by the immune system is rare because of immune suppression by the microenvironment.

[0099] The term “effective amount” or “therapeutically effective amount” refers to that amount of a compound or combination of compounds as described herein that is sufficient to effect the intended application including, but not limited to, disease treatment. A therapeutically effective amount may vary depending upon the intended application (in vitro or in vivo), or the subject and disease condition being treated (e.g., the weight, age and gender of the subject), the severity of the disease condition, or the manner of administration. The term also applies to a dose that will induce a particular response in target cells (e.g., the reduction of platelet adhesion and / or cell migration). The specific dose will vary depending on the particular compounds chosen, the dosing regimen to be followed, whether the compound is administered in combination with other compounds, timing of administration, the tissue to which it is administered, and the physical delivery system in which the compound is carried.

[0100] A “therapeutic effect” as that term is used herein, encompasses a therapeutic benefit and / or a prophylactic benefit. A prophylactic effect includes delaying or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof.

[0101] The terms “treatment”, “treating”, “treat”, and the like, refer to obtaining a desired pharmacologic and / or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or may be therapeutic in terms of a partial or complete cure for a disease and / or adverse effect attributable to the disease. “Treatment”, as used herein, covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development or progression; and (c) relieving the disease, i.e., causing regression of the disease and / or relieving one or more disease symptoms. “Treatment” is also meant to encompass delivery of an agent in order to provide for a pharmacologic effect, even in the absence of a disease or condition. For example, “treatment” encompasses delivery of a composition that can elicit an immune response or confer immunity in the absence of a disease condition, e.g., in the case of a vaccine.

[0102] The terms “non-myeloablative chemotherapy,”“non-myeloablative lymphodepletion,”“NMALD,”“NMA LD,”“NMA-LD,” and any variants of the foregoing, are used interchangeably to indicate a chemotherapeutic regimen designed to deplete the patient's lymphoid immune cells while avoiding depletion of the patient's myeloid immune cells. Typically, the patient receives a course of non-myeloablative chemotherapy prior to the administration of tumor infiltrating lymphocytes to the patient as described herein.

[0103] The terms “QD,”“qd,” or “q.d.” mean quaque die, once a day, or once daily. The terms “BID,”“bid,” or “b.i.d.” mean his in die, twice a day, or twice daily. The terms “TID,”“tid,” or “t.i.d.” mean ter in die, three times a day, or three times daily. The terms “QID,”“qid,” or “q.i.d.” mean quater in die, four times a day, or four times daily.

[0104] By “tumor infiltrating lymphocytes” or “TILs” herein is meant a population of cells originally obtained as white blood cells that have left the bloodstream of a subject and migrated into a tumor. TILs include, but are not limited to, CD8+ cytotoxic T cells (lymphocytes), Th1 and Th17 CD4+ T cells, natural killer cells, dendritic cells and M1 macrophages. TILs include both primary and secondary TILs. “Primary TILs” are those that are obtained from patient tissue samples as outlined herein (sometimes referred to as “freshly harvested”), and “secondary TILs” are any TIL cell populations that have been expanded or proliferated as discussed herein, including, but not limited to bulk TILs, expanded TILs (“REP TILs”) as well as “reREP TILs” as discussed herein.

[0105] TILs can generally be defined either biochemically, using cell surface markers, or functionally, by their ability to infiltrate tumors and effect treatment. TILs can be generally categorized by expressing one or more of the following biomarkers: CD4, CD8, TCR ap, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25. Additionally and alternatively, TILs can be functionally defined by their ability to infiltrate solid tumors upon reintroduction into a patient. TILS may further be characterized by potency—for example, TILS may be considered potent if, for example, interferon (IFN) release is greater than about 50 μg / mL, greater than about 100 μg / mL, greater than about 150 μg / mL, or greater than about 200 μg / mL.

[0106] By “cryopreserved TILs” (or cryopreserved MILs or PBLs) herein is meant that TILs, either primary, bulk, or expanded (REP TILs), are treated and stored in the range of about −150° C. to −60° C. General methods for cryopreservation are also described elsewhere herein, including in the Examples. For clarity, “cryopreserved TILs” are distinguishable from frozen tissue samples which may be used as a source of primary TILs.

[0107] By “thawed cryopreserved TILs” (or thawed MILs or PBLs) herein is meant a population of TILs that was previously cryopreserved and then treated to return to room temperature or higher, including but not limited to cell culture temperatures or temperatures wherein TILs may be administered to a patient.

[0108] By “population of cells” (including TILs) herein is meant a number of cells that share common traits. In general, populations generally range from 1×106 to 1×1010 in number, with different TIL populations comprising different numbers. For example, initial growth of primary TILs in the presence of IL-2 results in a population of bulk TILs of roughly 1×108 cells. REP expansion is generally done to provide populations of 1.5×109 to 1.5×1010 cells for infusion.

[0109] In general, TILs are initially obtained from a patient tumor sample (“primary TILs”) and then expanded into a larger population for further manipulation as described herein, optionally cryopreserved, restimulated as outlined herein and optionally evaluated for phenotype and metabolic parameters as an indication of TIL health.

[0110] In general, the harvested cell suspension is called a “primary cell population” or a “freshly harvested” cell population.

[0111] In general, as discussed herein, the TILs are initially prepared by obtaining a primary population of TILs from a tumor resected from a patient as discussed herein (the “primary cell population” or “first cell population”). This is followed with an initial bulk expansion utilizing a culturing of the cells with IL-2, forming a second population of cells (sometimes referred to herein as the “bulk TIL population” or “second population”).

[0112] The term “cytotoxic lymphocyte” includes cytotoxic T (CTL) cells (including CD8+ cytotoxic T lymphocytes and CD4+ T-helper lymphocytes), natural killer T (NKT) cells and natural killer (NK) cells. Cytotoxic lymphocytes can include, for example, peripheral blood-derived ap TCR-positive or γδ TCR-positive T cells activated by tumor associated antigens and / or transduced with tumor specific chimeric antigen receptors or T-cell receptors, and tumor-infiltrating lymphocytes (TILs).

[0113] The term “central memory T cell” refers to a subset of T cells that in the human are CD45RO+ and constitutively express CCR7 (CCR7h i) and CD62L (CD62 hi). The surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R), and IL-15R. Transcription factors for central memory T cells include BCL-6, BCL-6B, MBD2, and BMII. Central memory T cells primarily secret IL-2 and CD40L as effector molecules after TCR triggering. Central memory T cells are predominant in the CD4 compartment in blood, and in the human are proportionally enriched in lymph nodes and tonsils.

[0114] The term “effector memory T cell” refers to a subset of human or mammalian T cells that, like central memory T cells, are CD45R0+, but have lost the constitutive expression of CCR7 (CCR7lo) and are heterogeneous or low for CD62L expression (CD62Llo). The surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R), and IL-15R. Transcription factors for central memory T cells include BLIMP1. Effector memory T cells rapidly secret high levels of inflammatory cytokines following antigenic stimulation, including interferon-7, IL-4, and IL-5. Effector memory T cells are predominant in the CD8 compartment in blood, and in the human are proportionally enriched in the lung, liver, and gut. CD8+ effector memory T cells carry large amounts of perforin. The term “closed system” refers to a system that is closed to the outside environment. Any closed system appropriate for cell culture methods can be employed with the methods of the present invention. Closed systems include, for example, but are not limited to closed G-containers. Once a tumor segment is added to the closed system, the system is no opened to the outside environment until the TILs are ready to be administered to the patient.

[0115] In some embodiments, methods of the present disclosure further include a “pre-REP” stage in which tumor tissue or cells from tumor tissue are grown in standard lab media (including without limitation RPMI) and treated the with reagents such as irradiated feeder cells and anti-CD3 antibodies to achieve a desired effect, such as increase in the number of TILS and / or an enrichment of the population for cells containing desired cell surface markers or other structural, biochemical or functional features. The pre-REP stage may utilize lab grade reagents (under the assumption that the lab grade reagents get diluted out during a later REP stage), making it easier to incorporate alternative strategies for improving TIL production. Therefore, in some embodiments, the disclosed TLR agonist and / or peptide or peptidomimetics can be included in the culture medium during the pre-REP stage. The pre-REP culture can in some embodiments, include IL-2. The present invention is directed in preferred aspects to novel methods of augmenting REPs with one or more additional restimulation protocols, also referred to herein as a “restimulation Rapid Expansion Protocol” or “reREP”, which leads surprisingly to expanded memory T cell subsets, including the memory effector T cell subset, and / or to markes enhancement in the glycolytic respiration as compared to freshly harvested TILs or thawed cryopreserved TILs for the restimulated TILs (sometimes referred to herein as “reTILs”). That is, by using a reREP procedure on cyropreserved TILs, patients can receive highly metabolically active, healthy TILs, leading to more favorable outcomes.

[0116] When “an anti-tumor effective amount”, “an tumor-inhibiting effective amount”, or “therapeutic amount” is indicated, the precise amount of the compositions of the present invention to be administered can be determined by a physician with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject). It can generally be stated that a pharmaceutical composition comprising the genetically modified cytotoxic lymphocytes described herein may be administered at a dosage of 104 to 1011 cells / kg body weight (e.g., 10 to 106, 101 to 1010, 105 to 1011, 106 to 1010, 106 to 1011, 107 to 1011, 107 to 1010, 108 to 1011, 108 to 1010, 109 to 1011, or 109 to 1010 cells / kg body weight), including all integer values within those ranges. Genetically modified cytotoxic lymphocytes compositions may also be administered multiple times at these dosages. The genetically modified cytotoxic lymphocytes can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988). The optimal dosage and treatment regime for a particular patient can readily be determined by one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly.

[0117] For the avoidance of doubt, it is intended herein that particular features (for example integers, characteristics, values, uses, diseases, formulae, compounds or groups) described in conjunction with a particular aspect, embodiment or example of the invention are to be understood as applicable to any other aspect, embodiment or example described herein unless incompatible therewith. Thus such features may be used where appropriate in conjunction with any of the definition, claims or embodiments defined herein. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of the features and / or steps are mutually exclusive. The invention is not restricted to any details of any disclosed embodiments. The invention extends to any novel one, or novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

[0118] The terms “about” and “approximately” mean within a statistically meaningful range of a value. Such a range can be within an order of magnitude, preferably within 50%, more preferably within 20%, more preferably still within 10%, and even more preferably within 5% of a given value or range. The allowable variation encompassed by the terms “about” or “approximately” depends on the particular system under study, and can be readily appreciated by one of ordinary skill in the art. Moreover, as used herein, the terms “about” and “approximately” mean that dimensions, sizes, formulations, parameters, shapes and other quantities and characteristics are not and need not be exact, but may be approximate and / or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art. In general, a dimension, size, formulation, parameter, shape or other quantity or characteristic is “about” or “approximate” whether or not expressly stated to be such. It is noted that embodiments of very different sizes, shapes and dimensions may employ the described arrangements.

[0119] The transitional terms “comprising,”“consisting essentially of,” and “consisting of,” when used in the appended claims, in original and amended form, define the claim scope with respect to what unrecited additional claim elements or steps, if any, are excluded from the scope of the claim(s). The term “comprising” is intended to be inclusive or open-ended and does not exclude any additional, unrecited element, method, step or material. The term “consisting of” excludes any element, step or material other than those specified in the claim and, in the latter instance, impurities ordinary associated with the specified material(s). The term “consisting essentially of” limits the scope of a claim to the specified elements, steps or material(s) and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. All compositions, methods, and kits described herein that embody the present invention can, in alternate embodiments, be more specifically defined by any of the transitional terms “comprising,”“consisting essentially of,” and “consisting of.”Methods of Treating Cancers Including Pre-Treatment with ITK Inhibitors

[0120] The compositions and combinations of PBLs (and populations thereof) described above can be used in a method for treating hyperproliferative disorders. In a preferred embodiment, they are for use in treating cancers. In a preferred embodiment, the invention provides a method of treating a cancer, wherein the cancer is a hematological malignancy, such as a liquid tumor. In a preferred embodiment, the invention provides a method of treating a cancer, wherein the cancer is a hematological malignancy selected from the group consisting of acute myeloid leukemia (AML), AML without genetic markers or without molecular targets, MDS without genetic markers or without molecular targets, low grade MDS, hypoplastic MDS, CD123 negative (IL-3R negative) AML, FLT3 mutation negative AML, FLT3 positive AML, Nucleophosmin (NPM1) mutation negative acute myeloid leukemia, NPM1 positive AML, mantle cell lymphoma (MCL), follicular lymphoma (FL), diffuse large B cell lymphoma (DLBCL), activated B cell (ABC) DLBCL, germinal center B cell (GCB) DLBCL, chronic lymphocytic leukemia (CLL), CLL with Richter's transformation (or Richter's syndrome), CLL replased after treatment with BTK inhibitors, CLL relapsed after treatment with BCL2 inhibitors, CLL replased after treatment with BTK and BCL2 inhibitors, Relapsed CLL with 17p deletion, small lymphocytic leukemia (SLL), SLL replased after treatment with BTK inhibitors, SLL relapsed after treatment with BCL2 inhibitors, SLL replased after treatment with BTK and BCL2 inhibitors, Relapsed SLL with 17p deletion, CD19 negative B cell leukemia or lymphoma (including CLL / SLL), CD20 negative B cell leukemia or lymphoma (including CLL / SLL), CD23 negative B cell leukemia or lymphoma (including CLL / SLL), non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, relapsed and / or refractory Hodgkin's lymphoma, B cell acute lymphoblastic leukemia (B-ALL), mature B-ALL, Burkitt's lymphoma, Waldenström's macroglobulinemia (WM), multiple myeloma, myelodysplastic syndromes, myelofibrosis, chronic myelocytic leukemia, follicle center lymphoma, indolent NHL, human immunodeficiency virus (HIV) associated B cell lymphoma, Epstein-Barr virus (EBV) associated B cell lymphoma, EBV related lymphoproliferative disorders and post-transplant lymphoproliferative disorders, including subpopulations of patients with the foregoing diseases that are refractory to, intolerant to, or relapsed from treatment with a BTK inhibitor, including ibrutinib.

[0121] In an embodiment of the present invention, CLL patients who have been pretreated with ibrutinib represent a subpopulation of patients that can be successfully treated with the PBLs of the present invention. In particular, CLL patients who have been pretreated with ibrutinib, and who are no longer responsive to ibrutinib treatment, represent a subpopulation of patients that can be successfully treated with the PBLs of the present invention. In another embodiment, CLL patients who have been pretreated with ibrutinib and who have developed Richter's transformation (or Richter's syndrome), represent a subpopulation of patients that can be successfully treated with the PBLs of the present invention. In another embodiment, CLL patients who have been pretreated with ibrutinib, who have developed Richter's transformation (or Richter's syndrome) and who are no longer responsive to ibrutinib treatment, represent a subpopulation of patients that can be successfully treated with the PBLs of the present invention.

[0122] In some embodiments of the present invention, CLL patients who have relapsed after treatment with BTK+ / −BCL2 inhibitors represent a subpopulation of patients that can be successfully treated with the PBLs of the present invention. In some embodiments, CLL patients who are ineligible for CAR-T therapy represent a subpopulation of patients that can be successfully treated with the PBLs of the present invention. In some embodiments, CLL patients who have relapsed and have 17p deletion represent a subpopulation of patients that can be successfully treated with the PBLs of the present invention.

[0123] In some embodiments of the present invention, SLL patients who have relapsed after treatment with BTK+ / −BCL2 inhibitors represent a subpopulation of patients that can be successfully treated with the PBLs of the present invention. In some embodiments, SLL patients who are ineligible for CAR-T therapy represent a subpopulation of patients that can be successfully treated with the PBLs of the present invention. In some embodiments, SLL patients who have relapsed and have 17p deletion represent a subpopulation of patients that can be successfully treated with the PBLs of the present invention.

[0124] In some embodiments of the present invention, patients who have CD19 negative B cell leukemia or lymphoma including CLL / SLL represent a subpopulation of patients that can be successfully treated with the PBLs of the present invention. In some embodiments, patients who have CD20 negative B cell leukemia or lymphoma including CLL / SLL represent a subpopulation of patients that can be successfully treated with the PBLs of the present invention. In some embodiments, patients who have CD23 negative B cell leukemia or lymphoma including CLL / SLL represent a subpopulation of patients that can be successfully treated with the PBLs of the present invention.

[0125] In some embodiments, patients who have CD123 negative (IL-3R negative) AML represent a subpopulation of patients that can be successfully treated with the PBLs of the present invention. In some embodiments, patients who have FLT3 mutation negative (or FLT3 mutation positive) AML represent a subpopulation of patients that can be successfully treated with the PBLs of the present invention. In some embodiments, patients who have nucleophosmin (NPM1) mutation negative (or NPM1 mutation positive) AML represent a subpopulation of patients that can be successfully treated with the PBLs of the present invention. In some embodiments of the present invention, patients diagnosed with AML without specific genetic markers or without specific molecular targets represent a subpopulation of pateints that can be successfully treated with PBLs of the present invention.

[0126] In some embodiments of the present invention, patients diagnosed with myelodysplastic syndrome (MDS) without specific genetic markers or without specific molecular targets represent a subpopulation of pateints that can be successfully treated with PBLs of the present invention. In some embodiments, MDS patients with low grade MDS or hypoplastic MDS represent a subpopulation of pateints that can be successfully treated with PBLs of the present invention.

[0127] In an embodiment, the invention provides a method of treating a cancer, wherein the cancer is a hematological malignancy that responds to therapy with PD-1 and / or PD-L1 inhibitors including pembrolizumab, nivolumab, durvalumab, avelumab, or atezolizumab.

[0128] In an embodiment, the invention provides a method of treating a cancer in a patient with a population of PBLs comprising the steps of:

[0129] (a) obtaining peripheral blood mononuclear cells (PBMCs) from less than or equal to about 50 mL of whole blood from the patient, wherein the patient is optionally pretreated with an ITK inhibitor;

[0130] (b) admixing beads selective for CD3 and CD28 with the PBMCs, wherein the beads are added at a ratio of 3 beads:1 cell, to form an admixture of PBMCs and beads;

[0131] (c) culturing the admixture of PBMCs and beads at a density of about 25,000 cells per cm2 to about 50,000 cells per cm2 on a gas-permeable surface of one or more containers containing a first cell culture medium and IL-2 for a period of about 4 days;

[0132] (d) adding to each container of step (c) IL-2 and a second cell culture medium that is the same as or different from the first cell culture medium and culturing for a period of about 5 days to about 7 days to form an expanded population of PBLs;

[0133] (e) harvesting from each container the expanded population of PBLs;

[0134] (f) removing residual beads from the harvested population of PBLs to provide a PBL product;

[0135] (f) formulating and optionally cryopreserving the PBL product; and

[0136] (g) administering to the patient a therapeutically effective amount of the PBL product, wherein the ITK inhibitor is optionally an ITK inhibitor that covalently binds to ITK.

[0137] In an embodiment, the invention provides a method of treating a cancer in a patient with a population of PBLs comprising the steps of

[0138] (a) obtaining peripheral blood mononuclear cells (PBMCs) from less than or equal to about 50 mL of whole blood from the patient, wherein the patient is optionally pretreated with an ITK inhibitor;

[0139] (b) admixing beads selective for CD3 and CD28 with the PBMCs, wherein the beads are added at a ratio of 3 beads:1 cell, to form an admixture of PBMCs and beads;

[0140] (c) culturing the admixture of PBMCs and beads at a density of about 25,000 cells per cm2 to about 50,000 cells per cm2 on a gas-permeable surface of one or more containers containing a first cell culture medium and IL-2 for a period of about 4 days;

[0141] (d) adding to each container of step (c) IL-2 and a second cell culture medium that is the same as or different from the first cell culture medium and culturing for a period of about 5 days to about 7 days to form an expanded population of PBLs;

[0142] (e) harvesting from each container the expanded population of PBLs;

[0143] (f) removing residual beads from the harvested population of PBLs to provide a PBL product;

[0144] (i) formulating and optionally cryopreserving the PBL product; and

[0145] (j) administering to the patient a therapeutically effective amount of the PBL product, wherein the ITK inhibitor is optionally an ITK inhibitor that covalently binds to ITK, and wherein the cancer is a hematological malignancy selected from the group consisting of acute myeloid leukemia (AML), AML without genetic markers or without molecular targets, MDS without genetic markers or without molecular targets, low grade MDS, hypoplastic MDS, CD123 negative (IL-3R negative) AML, FLT3 mutation negative AML, FLT3 positive AML, Nucleophosmin (NPM1) mutation negative acute myeloid leukemia, NPM1 positive AML, mantle cell lymphoma (MCL), follicular lymphoma (FL), diffuse large B cell lymphoma (DLBCL), activated B cell (ABC) DLBCL, germinal center B cell (GCB) DLBCL, chronic lymphocytic leukemia (CLL), CLL with Richter's transformation (or Richter's syndrome), CLL replased after treatment with BTK inhibitors, CLL relapsed after treatment with BCL2 inhibitors, CLL replased after treatment with BTK and BCL2 inhibitors, Relapsed CLL with 17p deletion, small lymphocytic leukemia (SLL), SLL replased after treatment with BTK inhibitors, SLL relapsed after treatment with BCL2 inhibitors, SLL replased after treatment with BTK and BCL2 inhibitors, Relapsed SLL with 17p deletion, CD19 negative B cell leukemia or lymphoma (including CLL / SLL), CD20 negative B cell leukemia or lymphoma (including CLL / SLL), CD23 negative B cell leukemia or lymphoma (including CLL / SLL), non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, relapsed and / or refractory Hodgkin's lymphoma, B cell acute lymphoblastic leukemia (B-ALL), mature B-ALL, Burkitt's lymphoma, Waldenström's macroglobulinemia (WM), multiple myeloma, myelodysplastic syndromes, myelofibrosis, chronic myelocytic leukemia, follicle center lymphoma, indolent NHL, human immunodeficiency virus (HIV) associated B cell lymphoma, Epstein-Barr virus (EBV) associated B cell lymphoma, EBV related lymphoproliferative disorders and post-transplant lymphoproliferative disorders.

[0146] In an embodiment, the invention provides a method of treating a cancer in a patient with a population of PBLs comprising:

[0147] a. Obtaining a sample of peripheral blood mononuclear cells (PBMCs) from the peripheral blood of a patient, wherein said sample is optionally cryopreserved and the patient is optionally pretreated with an ITK inhibitor;

[0148] b. Optionally washing the PBMCs by centrifugation;

[0149] c. Admixing magnetic beads selective for CD3 and CD28 to the PBMCs to form an admixture of the beads and the PBMCs;

[0150] d. Seeding the admixture of the beads and the PBMCs into a gas-permeable container and co-culturing said PBMCs in media comprising about 3000 IU / mL of IL-2 in for about 4 to about 6 days;

[0151] e. Feeding said PBMCs using media comprising about 3000 IU / mL of IL-2, and co-culturing said PBMCs for about 5 days, such that the total co-culture period of steps d and e is about 9 to about 11 days;

[0152] f. Harvesting PBMCs from media;

[0153] g. Removing residual magnetic beads selective for CD3 and CD28 from the harvested PBMCs using a magnet;

[0154] h. Removing residual B-cells from the harvested PBMCs using magnetic-activated cell sorting and beads selective for CD19 to provide a PBL product;

[0155] i. Washing and concentrating the PBL product using a cell harvester;

[0156] j. Formulating and optionally cryopreserving the PBL product; and

[0157] k. Administering to the patient a therapeutically effective amount of the PBL product, wherein the ITK inhibitor is optionally an ITK inhibitor that covalently binds to ITK.

[0158] In an embodiment, the invention provides a method of treating a cancer in a patient with a population of PBLs comprising:

[0159] a. Obtaining a sample of peripheral blood mononuclear cells (PBMCs) from the peripheral blood of a patient, wherein said sample is optionally cryopreserved and the patient is optionally pretreated with an ITK inhibitor;

[0160] b. Optionally washing the PBMCs by centrifugation;

[0161] c. Admixing magnetic beads selective for CD3 and CD28 to the PBMCs to form an admixture of the beads and the PBMCs;

[0162] d. Seeding the admixture of the beads and the PBMCs into a gas-permeable container and co-culturing said PBMCs in media comprising about 3000 IU / mL of IL-2 in for about 4 to about 6 days;

[0163] e. Feeding said PBMCs using media comprising about 3000 IU / mL of IL-2, and co-culturing said PBMCs for about 5 days, such that the total co-culture period of steps d and e is about 9 to about 11 days;

[0164] f. Harvesting PBMCs from media;

[0165] g. Removing residual magnetic beads selective for CD3 and CD28 from the harvested PBMCs using a magnet;

[0166] h. Removing residual B-cells from the harvested PBMCs using magnetic-activated cell sorting and beads selective for CD19 to provide a PBL product;

[0167] i. Washing and concentrating the PBL product using a cell harvester;

[0168] j. Formulating and optionally cryopreserving the PBL product; and administering to the patient an effective amount of the PBL product, wherein the ITK inhibitor is optionally an ITK inhibitor that covalently binds to ITK, and wherein the cancer is a hematological malignancy selected from the group consisting of acute myeloid leukemia (AML), AML without genetic markers or without molecular targets, MDS without genetic markers or without molecular targets, low grade MDS, hypoplastic MDS, CD123 negative (IL-3R negative) AML, FLT3 mutation negative AML, FLT3 positive AML, Nucleophosmin (NPM1) mutation negative acute myeloid leukemia, NPM1 positive AML, mantle cell lymphoma (MCL), follicular lymphoma (FL), diffuse large B cell lymphoma (DLBCL), activated B cell (ABC) DLBCL, germinal center B cell (GCB) DLBCL, chronic lymphocytic leukemia (CLL), CLL with Richter's transformation (or Richter's syndrome), CLL replased after treatment with BTK inhibitors, CLL relapsed after treatment with BCL2 inhibitors, CLL replased after treatment with BTK and BCL2 inhibitors, Relapsed CLL with 17p deletion, small lymphocytic leukemia (SLL), SLL replased after treatment with BTK inhibitors, SLL relapsed after treatment with BCL2 inhibitors, SLL replased after treatment with BTK and BCL2 inhibitors, Relapsed SLL with 17p deletion, CD19 negative B cell leukemia or lymphoma (including CLL / SLL), CD20 negative B cell leukemia or lymphoma (including CLL / SLL), CD23 negative B cell leukemia or lymphoma (including CLL / SLL), non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, relapsed and / or refractory Hodgkin's lymphoma, B cell acute lymphoblastic leukemia (B-ALL), mature B-ALL, Burkitt's lymphoma, Waldenström's macroglobulinemia (WM), multiple myeloma, myelodysplastic syndromes, myelofibrosis, chronic myelocytic leukemia, follicle center lymphoma, indolent NHL, human immunodeficiency virus (HIV) associated B cell lymphoma, Epstein-Barr virus (EBV) associated B cell lymphoma, EBV related lymphoproliferative disorders and post-transplant lymphoproliferative disorders.

[0169] In an embodiment, the invention provides the method of treating cancer in a patient described in any of the preceding paragraphs as applicable above modified such that before the step of admixing beads selective for CD3 and CD28 with the PBMCs the method further comprises performing the step of removing B-cells from the PBMCs to provide PBMCs depleted of B-cells.

[0170] In an embodiment, the invention provides the method of treating cancer in a patient described in any of the preceding paragraphs as applicable above modified such that before the step of admixing beads selective for CD3 and CD28 with the PBMCs the method further comprises performing the steps of: (i) determining the proportion of the PMBCs constituted by B-cells as a B-cell percentage; and (ii) if the B-cell percentage determined in step (i) is at least about seventy percent (70%), removing B-cells from the PBMCs by selecting against CD19 to provide PBMCs depleted of B-cells.

[0171] In an embodiment, the invention provides any of the method of treating cancer in a patient described in any of the preceding paragraphs as applicable above modified such that if the B-cell percentage is at least about 75% the B-cell removal step is performed.

[0172] In an embodiment, the invention provides the method of treating cancer in a patient described in any of the preceding paragraphs as applicable above modified such that if the B-cell percentage is at least about 80% the B-cell removal step is performed.

[0173] In an embodiment, the invention provides the method of treating cancer in a patient described in any of the preceding paragraphs as applicable above modified such that if the B-cell percentage is at least about 85% the B-cell removal step is performed.

[0174] In an embodiment, the invention provides the method of treating cancer in a patient described in any of the preceding paragraphs as applicable above modified such that if the B-cell percentage is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% the B-cell removal step is performed.

[0175] In an embodiment of the invention, the invention provides the method of treating cancer in a patient described in any of the preceding paragraphs as applicable above modified such that removal of B-cells, or B-cell depletion (BCD), occurs on Day 0 or on Day 9 of a 9-day expansion process. In another embodiment, the BCD occurs on both Day 0 and Day 9 of a 9-day expansion process. In an embodiment of the invention, BCD occurs on Day 0 or Day 11 of an 11-day expansion process. In another embodiment, the BCD occurs on both Day 0 and Day 11 of an 11-day expansion process.

[0176] In an embodiment of the invention, the invention provides the method of treating cancer in a patient described in any of the preceding paragraphs as applicable above modified such that the BCD step is performed on a PBMC sample from a patient having a high initial B-cell count. In one embodiment, a high initial B-cell count is about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more B-cells in the initial PBMC sample.

[0177] In an embodiment, the invention provides the method of treating cancer in a patient described in any of the preceding paragraphs as applicable above modified such that the B-cell percentage is determined by comparison of the CD19+ cells to the CD45+ cells in the PBMCs.

[0178] In an embodiment, the invention provides the method of treating cancer in a patient described in any of the preceding paragraphs as applicable above modified such that the B-cell percentage is determined by comparison of the fraction of CD19+ / CD45+ cells to the fraction of CD45+ cells in the PBMCs.

[0179] In an embodiment, the invention provides the method of treating cancer in a patient described in any of the preceding paragraphs as applicable above modified such that the comparison of the fraction of CD19+ cells to the fraction of CD45+ cells in the PBMCs is performed by contacting the PBMCs with a CD19 stain and a CD45 stain, and then comparing the subpopulation of PBMCs positive for the both CD19 stain and the CD45 stain with the subpopulation of PBMCs positive for only the CD19 stain.

[0180] In an embodiment, the invention provides the method of treating cancer in a patient described in any of the preceding paragraphs as applicable above modified such that the CD19 stain is an anti-CD19 antibody conjugated to a first label and the CD45 stain is an anti-CD45 antibody conjugated to a second label.

[0181] In an embodiment, the invention provides the method of treating cancer in a patient described in any of the preceding paragraphs as applicable above modified such that the first label is a first fluorochrome and the second label is a second fluorochrome that is different from the first fluorochrome.

[0182] In an embodiment, the invention provides the method of treating cancer in a patient described in any of the preceding paragraphs as applicable above modified such that the step of removing B-cells from the PBMCs is performed by selecting against CD19 to provide PBMCs depleted of B-cells.

[0183] In an embodiment, the invention provides the method of treating cancer in a patient described in any of the preceding paragraphs as applicable above modified such that the step of removing B-cells from the PBMCs is performed by admixing beads selective for CD19 to the PBMCs to form complexes of the beads and CD19+ cells and removing the complexes from the PBMCs to provide PBMCs depleted of B-cells.

[0184] In an embodiment, the invention provides the method of treating cancer in a patient described in any of the preceding paragraphs as applicable above modified such that the step of removing B-cells from the PBMCs is performed by admixing magnetic beads selective for CD19 to the PBMCs to form complexes of the magnetic beads and CD19+ cells and using a magnet to remove the complexes from the PBMCs to provide PBMCs depleted of B-cells.

[0185] In an embodiment, the invention provides a pharmaceutical composition for use in a method of treating a cancer in a patient comprising the steps of:

[0186] (a) obtaining peripheral blood mononuclear cells (PBMCs) from less than or equal to about 50 mL of whole blood from the patient, wherein the patient is optionally pretreated with an ITK inhibitor;

[0187] (b) admixing beads selective for CD3 and CD28 with the PBMCs, wherein the beads are added at a ratio of 3 beads:1 cell, to form an admixture of PBMCs and beads;

[0188] (c) culturing the admixture of PBMCs and beads at a density of about 25,000 cells per cm2 to about 50,000 cells per cm2 on a gas-permeable surface of one or more containers containing a first cell culture medium and IL-2 for a period of about 4 days;

[0189] (d) adding to each container of step (c) IL-2 and a second cell culture medium that is the same as or different from the first cell culture medium and culturing for a period of about 5 days to about 7 days to form an expanded population of PBLs;

[0190] (e) harvesting from each container the expanded population of PBLs;

[0191] (f) removing residual beads from the harvested population of PBLs to provide a PBL product;

[0192] (h) formulating the PBL product to form a pharmaceutical composition and optionally cryopreserving the pharmaceutical composition; and

[0193] (i) administering to the patient a therapeutically effective amount of the pharmaceutical composition, wherein the ITK inhibitor is optionally an ITK inhibitor that covalently binds to ITK.

[0194] In an embodiment, the invention provides a pharmaceutical composition for use in a method of treating a cancer in a patient comprising the steps of:

[0195] (a) obtaining peripheral blood mononuclear cells (PBMCs) from less than or equal to about 50 mL of whole blood from the patient, wherein the patient is optionally pretreated with an ITK inhibitor;

[0196] (b) admixing beads selective for CD3 and CD28 with the PBMCs, wherein the beads are added at a ratio of 3 beads:1 cell, to form an admixture of PBMCs and beads;

[0197] (c) culturing the admixture of PBMCs and beads at a density of about 25,000 cells per cm2 to about 50,000 cells per cm2 on a gas-permeable surface of one or more containers containing a first cell culture medium and IL-2 for a period of about 4 days;

[0198] (d) adding to each container of step (c) IL-2 and a second cell culture medium that is the same as or different from the first cell culture medium and culturing for a period of about 5 days to about 7 days to form an expanded population of PBLs;

[0199] (e) harvesting from each container the expanded population of PBLs;

[0200] (f) removing residual beads from the harvested population of PBLs to provide a PBL product;

[0201] (g) formulating the PBL product to form a pharmaceutical composition and optionally cryopreserving the pharmaceutical composition; and administering to the patient a therapeutically effective amount of the pharmaceutical composition, wherein the ITK inhibitor is optionally an ITK inhibitor that covalently binds to ITK, and wherein the cancer is a hematological malignancy selected from the group consisting of acute myeloid leukemia (AML), AML without genetic markers or without molecular targets, MDS without genetic markers or without molecular targets, low grade MDS, hypoplastic MDS, CD123 negative (IL-3R negative) AML, FLT3 mutation negative AML, FLT3 positive AML, Nucleophosmin (NPM1) mutation negative acute myeloid leukemia, NPM1 positive AML, mantle cell lymphoma (MCL), follicular lymphoma (FL), diffuse large B cell lymphoma (DLBCL), activated B cell (ABC) DLBCL, germinal center B cell (GCB) DLBCL, chronic lymphocytic leukemia (CLL), CLL with Richter's transformation (or Richter's syndrome), CLL replased after treatment with BTK inhibitors, CLL relapsed after treatment with BCL2 inhibitors, CLL replased after treatment with BTK and BCL2 inhibitors, Relapsed CLL with 17p deletion, small lymphocytic leukemia (SLL), SLL replased after treatment with BTK inhibitors, SLL relapsed after treatment with BCL2 inhibitors, SLL replased after treatment with BTK and BCL2 inhibitors, Relapsed SLL with 17p deletion, CD19 negative B cell leukemia or lymphoma (including CLL / SLL), CD20 negative B cell leukemia or lymphoma (including CLL / SLL), CD23 negative B cell leukemia or lymphoma (including CLL / SLL), non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, relapsed and / or refractory Hodgkin's lymphoma, B cell acute lymphoblastic leukemia (B-ALL), mature B-ALL, Burkitt's lymphoma, Waldenström's macroglobulinemia (WM), multiple myeloma, myelodysplastic syndromes, myelofibrosis, chronic myelocytic leukemia, follicle center lymphoma, indolent NHL, human immunodeficiency virus (HIV) associated B cell lymphoma, Epstein-Barr virus (EBV) associated B cell lymphoma, EBV related lymphoproliferative disorders and post-transplant lymphoproliferative disorders.

[0202] In an embodiment, the invention provides a pharmaceutical composition for use in a method of treating a cancer in a patient comprising the steps of:

[0203] (a) Obtaining a sample of peripheral blood mononuclear cells (PBMCs) from the peripheral blood of a patient, wherein said sample is optionally cryopreserved and the patient is optionally pretreated with an ITK inhibitor;

[0204] (b) Optionally washing the PBMCs by centrifugation;

[0205] (c) Admixing magnetic beads selective for CD3 and CD28 to the PBMCs to form an admixture of the beads and the PBMCs;

[0206] (d) Seeding the admixture of the beads and the PBMCs into a gas-permeable container and co-culturing said PBMCs in media comprising about 3000 IU / mL of IL-2 in for about 4 to about 6 days;

[0207] (e) Feeding said PBMCs using media comprising about 3000 IU / mL of IL-2, and co-culturing said PBMCs for about 5 days, such that the total co-culture period of steps d and e is about 9 to about 11 days;

[0208] (f) Harvesting PBMCs from media;

[0209] (g) Removing residual magnetic beads selective for CD3 and CD28 from the harvested PBMCs using a magnet;

[0210] (h) Removing residual B-cells from the harvested PBMCs using magnetic-activated cell sorting and beads selective for CD19 to provide a PBL product;

[0211] (i) Washing and concentrating the PBL product using a cell harvester;

[0212] (j) Formulating the PBL product to form a pharmaceutical composition and optionally cryopreserving the pharmaceutical composition; and

[0213] (k) Administering to the patient a therapeutically effective amount of the pharmaceutical composition, wherein the ITK inhibitor is optionally an ITK inhibitor that covalently binds to ITK.

[0214] In an embodiment, the invention provides a pharmaceutical composition for use in a method of treating a cancer in a patient comprising the steps of:

[0215] (b) Obtaining a sample of peripheral blood mononuclear cells (PBMCs) from the peripheral blood of a patient, wherein said sample is optionally cryopreserved and the patient is optionally pretreated with an ITK inhibitor;

[0216] (b) Optionally washing the PBMCs by centrifugation,

[0217] (c) Admixing magnetic beads selective for CD3 and CD28 to the PBMCs to form an admixture of the beads and the PBMCs;

[0218] (d) Seeding the admixture of the beads and the PBMCs into a gas-permeable container and co-culturing said PBMCs in media comprising about 3000 IU / mL of IL-2 in for about 4 to about 6 days;

[0219] (e) Feeding said PBMCs using media comprising about 3000 IU / mL of IL-2, and co-culturing said PBMCs for about 5 days, such that the total co-culture period of steps d and e is about 9 to about 11 days;

[0220] (f) Harvesting PBMCs from media;

[0221] (g) Removing residual magnetic beads selective for CD3 and CD28 from the harvested PBMCs using a magnet;

[0222] (h) Removing residual B-cells from the harvested PBMCs using magnetic-activated cell sorting and beads selective for CD19 to provide a PBL product;

[0223] (i) Washing and concentrating the PBL product using a cell harvester;

[0224] (j) Formulating the PBL product to form a pharmaceutical composition and optionally cryopreserving the pharmaceutical composition; and

[0225] (k) Administering to the patient a therapeutically effective amount of the pharmaceutical composition, wherein the ITK inhibitor is optionally an ITK inhibitor that covalently binds to ITK, and wherein the cancer is a hematological malignancy selected from the group consisting of acute myeloid leukemia (AML), AML without genetic markers or without molecular targets, MDS without genetic markers or without molecular targets, low grade MDS, hypoplastic MDS, CD123 negative (IL-3R negative) AML, FLT3 mutation negative AML, FLT3 positive AML, Nucleophosmin (NPM1) mutation negative acute myeloid leukemia, NPM1 positive AML, mantle cell lymphoma (MCL), follicular lymphoma (FL), diffuse large B cell lymphoma (DLBCL), activated B cell (ABC) DLBCL, germinal center B cell (GCB) DLBCL, chronic lymphocytic leukemia (CLL), CLL with Richter's transformation (or Richter's syndrome), CLL replased after treatment with BTK inhibitors, CLL relapsed after treatment with BCL2 inhibitors, CLL replased after treatment with BTK and BCL2 inhibitors, Relapsed CLL with 17p deletion, small lymphocytic leukemia (SLL), SLL replased after treatment with BTK inhibitors, SLL relapsed after treatment with BCL2 inhibitors, SLL replased after treatment with BTK and BCL2 inhibitors, Relapsed SLL with 17p deletion, CD19 negative B cell leukemia or lymphoma (including CLL / SLL), CD20 negative B cell leukemia or lymphoma (including CLL / SLL), CD23 negative B cell leukemia or lymphoma (including CLL / SLL), non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, relapsed and / or refractory Hodgkin's lymphoma, B cell acute lymphoblastic leukemia (B-ALL), mature B-ALL, Burkitt's lymphoma, Waldenström's macroglobulinemia (WM), multiple myeloma, myelodysplastic syndromes, myelofibrosis, chronic myelocytic leukemia, follicle center lymphoma, indolent NHL, human immunodeficiency virus (HIV) associated B cell lymphoma, Epstein-Barr virus (EBV) associated B cell lymphoma, EBV related lymphoproliferative disorders and post-transplant lymphoproliferative disorders.

[0226] In an embodiment, the invention provides the pharmaceutical composition for use in a method of treating a cancer in a patient described in any of the preceding paragraphs as applicable above modified such that before the step of admixing beads selective for CD3 and CD28 with the PBMCs the method further comprises performing the step of removing B-cells from the PBMCs to provide PBMCs depleted of B-cells.

[0227] In an embodiment, the invention provides the pharmaceutical composition for use in a method of treating a cancer in a patient described in any of the preceding paragraphs as applicable above modified such that before the step of admixing beads selective for CD3 and CD28 with the PBMCs the method further comprises performing the steps of: (i) determining the proportion of the PMBCs constituted by B-cells as a B-cell percentage; and (ii) if the B-cell percentage determined in step (i) is at least about seventy percent (70%), removing B-cells from the PBMCs by selecting against CD19 to provide PBMCs depleted of B-cells.

[0228] In an embodiment, the invention provides the pharmaceutical composition for use in a method of treating a cancer in a patient described in any of the preceding paragraphs as applicable above modified such that if the B-cell percentage is at least about 75% the B-cell removal step is performed.

[0229] In an embodiment, the invention provides the pharmaceutical composition for use in a method of treating a cancer in a patient described in any of the preceding paragraphs as applicable above modified such that if the B-cell percentage is at least about 80% the B-cell removal step is performed.

[0230] In an embodiment, the invention provides the pharmaceutical composition for use in a method of treating a cancer in a patient described in any of the preceding paragraphs as applicable above modified such that if the B-cell percentage is at least about 85% the B-cell removal step is performed.

[0231] In an embodiment, the invention provides the pharmaceutical composition for use in a method of treating a cancer in a patient described in any of the preceding paragraphs as applicable above modified such that if the B-cell percentage is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% the B-cell removal step is performed.

[0232] In an embodiment of the invention, the invention provides the pharmaceutical composition for use in a method of treating a cancer in a patient described in any of the preceding paragraphs as applicable above modified such that removal of B-cells, or B-cell depletion (BCD), occurs on Day 0 or on Day 9 of a 9-day expansion process. In another embodiment, the BCD occurs on both Day 0 and Day 9 of a 9-day expansion process. In an embodiment of the invention, BCD occurs on Day 0 or Day 11 of an 11-day expansion process. In another embodiment, the BCD occurs on both Day 0 and Day 11 of an 11-day expansion process.

[0233] In an embodiment of the invention, the invention provides the pharmaceutical composition for use in a method of treating a cancer in a patient described in any of the preceding paragraphs as applicable above modified such that the BCD step is performed on a PBMC sample from a patient having a high initial B-cell count. In one embodiment, a high initial B-cell count is about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more B-cells in the initial PBMC sample.

[0234] In an embodiment, the invention provides the pharmaceutical composition for use in a method of treating a cancer in a patient described in any of the preceding paragraphs as applicable above modified such that the B-cell percentage is determined by comparison of the CD19+ cells to the CD45+ cells in the PBMCs.

[0235] In an embodiment, the invention provides the pharmaceutical composition for use in a method of treating a cancer in a patient described in any of the preceding paragraphs as applicable above modified such that the B-cell percentage is determined by comparison of the fraction of CD19+ / CD45+ cells to the fraction of CD45+ cells in the PBMCs.

[0236] In an embodiment, the invention provides the pharmaceutical composition for use in a method of treating a cancer in a patient described in any of the preceding paragraphs as applicable above modified such that the comparison of the fraction of CD19+ cells to the fraction of CD45+ cells in the PBMCs is performed by contacting the PBMCs with a CD19 stain and a CD45 stain, and then comparing the subpopulation of PBMCs positive for the both CD19 stain and the CD45 stain with the subpopulation of PBMCs positive for only the CD19 stain.

[0237] In an embodiment, the invention provides the pharmaceutical composition for use in a method of treating a cancer in a patient described in any of the preceding paragraphs as applicable above modified such that the CD19 stain is an anti-CD19 antibody conjugated to a first label and the CD45 stain is an anti-CD45 antibody conjugated to a second label.

[0238] In an embodiment, the invention provides the pharmaceutical composition for use in a method of treating a cancer in a patient described in any of the preceding paragraphs as applicable above modified such that the first label is a first fluorochrome and the second label is a second fluorochrome that is different from the first fluorochrome.

[0239] In an embodiment, the invention provides the pharmaceutical composition for use in a method of treating a cancer in a patient described in any of the preceding paragraphs as applicable above modified such that the step of removing B-cells from the PBMCs is performed by selecting against CD19 to provide PBMCs depleted of B-cells.

[0240] In an embodiment, the invention provides the pharmaceutical composition for use in a method of treating a cancer in a patient described in any of the preceding paragraphs as applicable above modified such that the step of removing B-cells from the PBMCs is performed by admixing beads selective for CD19 to the PBMCs to form complexes of the beads and CD19+ cells and removing the complexes from the PBMCs to provide PBMCs depleted of B-cells.

[0241] In an embodiment, the invention provides the pharmaceutical composition for use in a method of treating a cancer in a patient described in any of the preceding paragraphs as applicable above modified such that the step of removing B-cells from the PBMCs is performed by admixing magnetic beads selective for CD19 to the PBMCs to form complexes of the magnetic beads and CD19+ cells and using a magnet to remove the complexes from the PBMCs to provide PBMCs depleted of B-cells.

[0242] In an embodiment, the invention provides the use of a pharmaceutical composition in a method for the treatment of a cancer in a patient, the method comprising the steps of:

[0243] (a) obtaining peripheral blood mononuclear cells (PBMCs) from less than or equal to about 50 mL of whole blood from the patient, wherein the patient is optionally pretreated with an ITK inhibitor;

[0244] (b) admixing beads selective for CD3 and CD28 with the PBMCs, wherein the beads are added at a ratio of 3 beads:1 cell, to form an admixture of PBMCs and beads;

[0245] (c) culturing the admixture of PBMCs and beads at a density of about 25,000 cells per cm2 to about 50,000 cells per cm2 on a gas-permeable surface of one or more containers containing a first cell culture medium and IL-2 for a period of about 4 days;

[0246] (d) adding to each container of step (c) IL-2 and a second cell culture medium that is the same as or different from the first cell culture medium and culturing for a period of about 5 days to about 7 days to form an expanded population of PBLs;

[0247] (e) harvesting from each container the expanded population of PBLs;

[0248] (f) removing the beads from the harvested population of PBLs to provide a PBL product;

[0249] (g) formulating the PBL product to form a pharmaceutical composition and optionally cryopreserving the pharmaceutical composition; and

[0250] (h) administering to the patient a therapeutically effective amount of the pharmaceutical composition, wherein the ITK inhibitor is optionally an ITK inhibitor that covalently binds to ITK.

[0251] In an embodiment, the invention provides the use of a pharmaceutical composition in a method for the treatment of a cancer in a patient, the method comprising the steps of:

[0252] (a) obtaining peripheral blood mononuclear cells (PBMCs) from less than or equal to about 50 mL of whole blood from the patient, wherein the patient is optionally pretreated with an ITK inhibitor;

[0253] (b) admixing beads selective for CD3 and CD28 with the PBMCs, wherein the beads are added at a ratio of 3 beads:1 cell, to form an admixture of PBMCs and beads;

[0254] (c) culturing the admixture of PBMCs and beads at a density of about 25,000 cells per cm2 to about 50,000 cells per cm2 on a gas-permeable surface of one or more containers containing a first cell culture medium and IL-2 for a period of about 4 days;

[0255] (d) adding to each container of step (c) IL-2 and a second cell culture medium that is the same as or different from the first cell culture medium and culturing for a period of about 5 days to about 7 days to form an expanded population of PBLs;

[0256] (e) harvesting from each container the expanded population of PBLs;

[0257] (f) removing the beads from the harvested population of PBLs to provide a PBL product;

[0258] (g) formulating the PBL product to form a pharmaceutical composition and optionally cryopreserving the pharmaceutical composition; and

[0259] (h) administering to the patient a therapeutically effective amount of the pharmaceutical composition,wherein the ITK inhibitor is optionally an ITK inhibitor that covalently binds to ITK, and wherein the cancer is a hematological malignancy selected from the group consisting of acute myeloid leukemia (AML), AML without genetic markers or without molecular targets, MDS without genetic markers or without molecular targets, low grade MDS, hypoplastic MDS, CD123 negative (IL-3R negative) AML, FLT3 mutation negative AML, FLT3 positive AML, Nucleophosmin (NPM1) mutation negative acute myeloid leukemia, NPM1 positive AML, mantle cell lymphoma (MCL), follicular lymphoma (FL), diffuse large B cell lymphoma (DLBCL), activated B cell (ABC) DLBCL, germinal center B cell (GCB) DLBCL, chronic lymphocytic leukemia (CLL), CLL with Richter's transformation (or Richter's syndrome), CLL replased after treatment with BTK inhibitors, CLL relapsed after treatment with BCL2 inhibitors, CLL replased after treatment with BTK and BCL2 inhibitors, Relapsed CLL with 17p deletion, small lymphocytic leukemia (SLL), SLL replased after treatment with BTK inhibitors, SLL relapsed after treatment with BCL2 inhibitors, SLL replased after treatment with BTK and BCL2 inhibitors, Relapsed SLL with 17p deletion, CD19 negative B cell leukemia or lymphoma (including CLL / SLL), CD20 negative B cell leukemia or lymphoma (including CLL / SLL), CD23 negative B cell leukemia or lymphoma (including CLL / SLL), non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, relapsed and / or refractory Hodgkin's lymphoma, B cell acute lymphoblastic leukemia (B-ALL), mature B-ALL, Burkitt's lymphoma, Waldenström's macroglobulinemia (WM), multiple myeloma, myelodysplastic syndromes, myelofibrosis, chronic myelocytic leukemia, follicle center lymphoma, indolent NHL, human immunodeficiency virus (HIV) associated B cell lymphoma, Epstein-Barr virus (EBV) associated B cell lymphoma, EBV related lymphoproliferative disorders and post-transplant lymphoproliferative disorders.

[0260] In an embodiment, the invention provides the use of a pharmaceutical composition in a method of treating cancer in a patient, the method comprising the steps of:

[0261] (a) Obtaining a sample of peripheral blood mononuclear cells (PBMCs) from the peripheral blood of a patient, wherein said sample is optionally cryopreserved and the patient is optionally pretreated with an ITK inhibitor;

[0262] (b) Optionally washing the PBMCs by centrifugation;

[0263] (c) Adding magnetic beads selective for CD3 and CD28 to the PBMCs;

[0264] (d) Seeding PBMCs into a gas-permeable container and co-culturing said PBMCs in media comprising about 3000 IU / mL of IL-2 in for about 4 to about 6 days;

[0265] (e) Feeding said PBMCs using media comprising about 3000 IU / mL of IL-2, and co-culturing said PBMCs for about 5 days, such that the total co-culture period of steps d and e is about 9 to about 11 days;

[0266] (f) Harvesting PBMCs from media;

[0267] (g) Removing residual magnetic beads selective for CD3 and CD28 from the harvested PBMCs using a magnet;

[0268] (h) Removing residual B-cells from the harvested PBMCs using magnetic-activated cell sorting and CD19′ beads to provide a PBL product;

[0269] (i) Washing and concentrating the PBL product using a cell harvester;

[0270] (j) Formulating the PBL product to form a pharmaceutical composition and optionally cryopreserving the pharmaceutical composition; and

[0271] (k) Administering to the patient a therapeutically effective amount of the pharmaceutical composition, wherein the ITK inhibitor is optionally an ITK inhibitor that covalently binds to ITK.

[0272] In an embodiment, the invention provides the use of a pharmaceutical composition in a method of treating cancer in a patient, the method comprising the steps of:

[0273] (a) Obtaining a sample of peripheral blood mononuclear cells (PBMCs) from the peripheral blood of a patient, wherein said sample is optionally cryopreserved and the patient is optionally pretreated with an ITK inhibitor;

[0274] (b) Optionally washing the PBMCs by centrifugation;

[0275] (c) Adding magnetic beads selective for CD3 and CD28 to the PBMCs;

[0276] (d) Seeding PBMCs into a gas-permeable container and co-culturing said PBMCs in media comprising about 3000 IU / mL of IL-2 in for about 4 to about 6 days;

[0277] (e) Feeding said PBMCs using media comprising about 3000 IU / mL of IL-2, and co-culturing said PBMCs for about 5 days, such that the total co-culture period of steps d and e is about 9 to about 11 days;

[0278] (f) Harvesting PBMCs from media;

[0279] (g) Removing the magnetic beads selective for CD3 and CD28 using a magnet;

[0280] (h) Removing residual B-cells using magnetic-activated cell sorting and CD19+ beads to provide a PBL product;

[0281] (i) Washing and concentrating the PBL product using a cell harvester;

[0282] (j) Formulating the PBL product to form a pharmaceutical composition and optionally cryopreserving the pharmaceutical composition; and

[0283] (k) Administering to the patient a therapeutically effective amount of the pharmaceutical composition, wherein the ITK inhibitor is optionally an ITK inhibitor that covalently binds to ITK, and wherein the cancer is a hematological malignancy selected from the group consisting of acute myeloid leukemia (AML), AML without genetic markers or without molecular targets, MDS without genetic markers or without molecular targets, low grade MDS, hypoplastic MDS, CD123 negative (IL-3R negative) AML, FLT3 mutation negative AML, FLT3 positive AML, Nucleophosmin (NPM1) mutation negative acute myeloid leukemia, NPM1 positive AML, mantle cell lymphoma (MCL), follicular lymphoma (FL), diffuse large B cell lymphoma (DLBCL), activated B cell (ABC) DLBCL, germinal center B cell (GCB) DLBCL, chronic lymphocytic leukemia (CLL), CLL with Richter's transformation (or Richter's syndrome), CLL replased after treatment with BTK inhibitors, CLL relapsed after treatment with BCL2 inhibitors, CLL replased after treatment with BTK and BCL2 inhibitors, Relapsed CLL with 17p deletion, small lymphocytic leukemia (SLL), SLL replased after treatment with BTK inhibitors, SLL relapsed after treatment with BCL2 inhibitors, SLL replased after treatment with BTK and BCL2 inhibitors, Relapsed SLL with 17p deletion, CD19 negative B cell leukemia or lymphoma (including CLL / SLL), CD20 negative B cell leukemia or lymphoma (including CLL / SLL), CD23 negative B cell leukemia or lymphoma (including CLL / SLL), non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, relapsed and / or refractory Hodgkin's lymphoma, B cell acute lymphoblastic leukemia (B-ALL), mature B-ALL, Burkitt's lymphoma, Waldenström's macroglobulinemia (WM), multiple myeloma, myelodysplastic syndromes, myelofibrosis, chronic myelocytic leukemia, follicle center lymphoma, indolent NHL, human immunodeficiency virus (HIV) associated B cell lymphoma, Epstein-Barr virus (EBV) associated B cell lymphoma, EBV related lymphoproliferative disorders and post-transplant lymphoproliferative disorders.

[0284] In an embodiment, the invention provides the use of a pharmaceutical composition in a method of treating cancer in a patient described in any of the preceding paragraphs as applicable above modified such that before the step of admixing beads selective for CD3 and CD28 with the PBMCs the method further comprises performing the step of removing B-cells from the PBMCs to provide PBMCs depleted of B-cells.

[0285] In an embodiment, the invention provides the use of a pharmaceutical composition in a method of treating cancer in a patient described in any of the preceding paragraphs as applicable above modified such that before the step of admixing beads selective for CD3 and CD28 with the PBMCs the method further comprises performing the steps of: (i) determining the proportion of the PMBCs constituted by B-cells as a B-cell percentage; and (ii) if the B-cell percentage determined in step (i) is at least about seventy percent (70%), removing B-cells from the PBMCs by selecting against CD19 to provide PBMCs depleted of B-cells.

[0286] In an embodiment, the invention provides the use of a pharmaceutical composition in a method of treating cancer in a patient described in any of the preceding paragraphs as applicable above modified such that if the B-cell percentage is at least about 75% the B-cell removal step is performed.

[0287] In an embodiment, the invention provides the use of a pharmaceutical composition in a method of treating cancer in a patient described in any of the preceding paragraphs as applicable above modified such that if the B-cell percentage is at least about 80% the B-cell removal step is performed.

[0288] In an embodiment, the invention provides the use of a pharmaceutical composition in a method of treating cancer in a patient described in any of the preceding paragraphs as applicable above modified such that if the B-cell percentage is at least about 85% the B-cell removal step is performed.

[0289] In an embodiment, the invention provides the use of a pharmaceutical composition in a method of treating cancer in a patient described in any of the preceding paragraphs as applicable above modified such that if the B-cell percentage is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% the B-cell removal step is performed.

[0290] In an embodiment, the invention provides the use of a pharmaceutical composition in a method of treating cancer in a patient described in any of the preceding paragraphs as applicable above modified such that the B-cell percentage is determined by comparison of the CD19+ cells to the CD45+ cells in the PBMCs.

[0291] In an embodiment of the invention, the invention provides the use of a pharmaceutical composition in a method of treating cancer in a patient described in any of the preceding paragraphs as applicable above modified such that removal of B-cells, or B-cell depletion (BCD), occurs on Day 0 or on Day 9 of a 9-day expansion process. In another embodiment, the BCD occurs on both Day 0 and Day 9 of a 9-day expansion process. In an embodiment of the invention, BCD occurs on Day 0 or Day 11 of an 11-day expansion process. In another embodiment, the BCD occurs on both Day 0 and Day 11 of an 11-day expansion process.

[0292] In an embodiment of the invention, the invention provides the use of a pharmaceutical composition in a method of treating cancer in a patient described in any of the preceding paragraphs as applicable above modified such that the BCD step is performed on a PBMC sample from a patient having a high initial B-cell count. In one embodiment, a high initial B-cell count is about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more B-cells in the initial PBMC sample.

[0293] In an embodiment, the invention provides the use of a pharmaceutical composition in a method of treating cancer in a patient described in any of the preceding paragraphs as applicable above modified such that the B-cell percentage is determined by comparison of the fraction of CD19+ / CD45+ cells to the fraction of CD45+ cells in the PBMCs.

[0294] In an embodiment, the invention provides the use of a pharmaceutical composition in a method of treating cancer in a patient described in any of the preceding paragraphs as applicable above modified such that the comparison of the fraction of CD19+ cells to the fraction of CD45+ cells in the PBMCs is performed by contacting the PBMCs with a CD19 stain and a CD45 stain, and then comparing the subpopulation of PBMCs positive for the both CD19 stain and the CD45 stain with the subpopulation of PBMCs positive for only the CD19 stain.

[0295] In an embodiment, the invention provides the use of a pharmaceutical composition in a method of treating cancer in a patient described in any of the preceding paragraphs as applicable above modified such that the CD19 stain is an anti-CD19 antibody conjugated to a first label and the CD45 stain is an anti-CD45 antibody conjugated to a second label.

[0296] In an embodiment, the invention provides the use of a pharmaceutical composition in a method of treating cancer in a patient described in any of the preceding paragraphs as applicable above modified such that the first label is a first fluorochrome and the second label is a second fluorochrome that is different from the first fluorochrome.

[0297] In an embodiment, the invention provides the use of a pharmaceutical composition in a method of treating cancer in a patient described in any of the preceding paragraphs as applicable above modified such that before the step of admixing beads selective for CD3 and CD28 with the PBMCs the method further comprises performing the step of removing B-cells from the PBMCs by selecting against CD19 to provide PBMCs depleted of B-cells.

[0298] In an embodiment, the invention provides the use of a pharmaceutical composition in a method of treating cancer in a patient described in any of the preceding paragraphs as applicable above modified such that before the step of admixing beads selective for CD3 and CD28 with the PBMCs the method further comprises performing the step of removing B-cells from the PBMCs by admixing beads selective for CD19 with the PBMCs to form complexes of the beads and CD19+ cells in an admixture and removing the complexes from the admixture to provide PBMCs depleted of B-cells.

[0299] In an embodiment, the invention provides the use of a pharmaceutical composition in a method of treating cancer in a patient described in any of the preceding paragraphs as applicable above modified such that before the step of admixing beads selective for CD3 and CD28 with the PBMCs the method further comprises performing the step of removing B-cells from the PBMCs by admixing magnetic beads selective for CD19 with the PBMCs to form complexes of the magnetic beads and CD19+ cells in an admixture and using a magnet to remove the complexes from the admixture to provide PBMCs depleted of B-cells.

[0300] In any of the foregoing embodiments of the invention, pre-treatment with a kinase inhibitor is described. In an embodiment, the kinase inhibitor is selected from the group consisting of imatinib, dasatinib, ibrutinib, bosutinib, nilotinib, erlotinib, acalabrutinib, zanubrutinib, tirabrutinib, orelabrutinib, evobrutinib, spebrutinib, remibrutinib, tolebrutinib, olmutinib, branebrutinib, TAK-020, elsubrutinib, rilzabrutinib, vecabrutinib, fenebrutinib, GNE-431, RN-486, BMS935177, BMS-986142, CGI-1746, GDC-0834, G-744, G-278, or other kinase inhibitors, tyrosine kinase inhibitors, serine / threonine kinase inhibitors, or BTK inhibitors known in the art. In an embodiment, pre-treatment regimens with a kinase inhibitor are as known in the art and / or as prescribed by a physician.

[0301] In any of the foregoing embodiments of the invention, pre-treatment with a BCL2 inhibitor is described. In an embodiment, the BCL2 inhibitor is selected from the group consisting of venetoclax, obatoclax, subatoclax, maritoclax, gossypol, apogossypol, TW-37, UMI-77, BDA-366, navitoclax, and ABT-737 or any other BCL2 inhibitor known in the art.

[0302] In any of the foregoing embodiments of the invention, pre-treatment with an IL-2-inducible T-cell kinase (ITK) inhibitor is described. Interleukin-2-inducible T cell kinase (ITK) is a non-receptor tyrosine kinase expressed in T-cells and regulates various pathways. Any ITK inhibitor known in the art may be used in embodiments of the present invention (see, for example, Lo, et al., Expert Opinion on Therapeutic Patents, 20:459-469 (2010); Vargas, et al., Scandinavian Journal of Immunology, 78(2):130-139 (2013); WO2015112847; WO2016118951; WO2007136790, US20120058984A1, and U.S. Pat. Nos. 9,531,689 and 9,695,200; all of which are incorporated by reference herein in their entireties). In an embodiment of the invention, the ITK inhibitor is a covalent JTK inhibitor that covalently and irreversibly binds to ITK. In an embodiment of the invention, the ITK inhibitor is an allosteric ITK inhibitor that binds to ITK. In an embodiment of the invention, the ITK inhibitor is selected from the group consisting of aminothiazole-based ITK inhibitors, 5-aminomethylbenzimdazoles-based ITK inhibitors, 3-Aminopyrid-2-ones-based ITK inhibitors, (4 or 5-aryl)pyrazolyl-indole-based ITK inhibitors, benzimidazole-based ITK inhibitors, aminobenzimidazole-based ITK inhibitors, aminopyrimidine-based ITK inhibitors, aminopyridine-based ITK inhibitors, diazolodiazine-based ITK inhibitors, triazole-based ITK inhibitors, 3-aminopyride-2-ones-based ITK inhibitors, indolylindazole-based ITK inhibitors, indole-based ITK inhibitors, aza-indole-based ITK inhibitors, pyrazolyl-indole-based inhibitors, thienopyrazole-based ITK inhibitors, heterocyclic ITK inhibitors, and ITK inhibitors targeting cysteine-442 in the ATP pocket (such as ibrutinib), aza-benzimidazole-based ITK inhibitors, benzothiazole-based ITK inhibitors, indole-based ITK inhibitors, pyridone-based ITK inhibitors, sulfoximine-substituted pyrimidine ITK inhibitors, arylpyridinone-based ITK inhibitors, and any other ITK inhibitors known in the art. In an embodiment of the invention, pre-treatment regimens with an ITK inhibitor are as known in the art and / or as prescribed by a physician. In an embodiment of the invention, the ITK inhibitor is selected from the group consisting ofand combinations thereof. In an embodiment of the invention, the ITK inhibitor is selected from the group consisting of imatinib, dasatinib (BMS-354825), Sprycel [N-(2-chloro-6-methylphenyl)-2-(6-(4-(2-hydroxyethyl)-piperazin-1-yl)-2-meth-ylpyrimidin-4-ylamino)thiazole-5-carboxamide), ibrutinib ((1-{(3R)-3-[4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]piperidin-1-yl}prop-2-en-1-one), bosutinib, nilotinib, erlotinib, 1H-pyrazolo[4,3-c]cinnolin-3-ol, CTA056 (7-benzyl-1-(3-(piperidin-1-yl)propyl)-2-(4-(pyridin-4-yl)phenyl)-1H-imidazo[4,5-g]quinoxalin-6(5H)-one), Compound 10 (Boehringer Ingelheim from Moriarty, et al., Bioorg Med Chem Lett, 18:5537-40 (2008)), Compound 19 (Boehringer Ingelheim from Moriarty, et al., Bioorg Med Chem Lett., 18:5537-40 (2008)), Compound 27 (Boehringer Ingelheim from Moriarty, et al., Bioorg Med Chem Lett., 18:5537-40 (2008)), Compound 26 (Boehringer Ingelheim from Winters, et al., Bioorg Med Chem Lett., 18:5541-4 (2008)), Compound 37 (Boehringer Ingelheim from Cook, et al., Bioorg Med Chem Lett., 19:773-7 (2009)), Compound 41 (Boehringer Ingelheim from Cook, et al., Bioorg Med Chem Lett., 19:773-7 (2009)), Compound 48 (Boehringer Ingelheim from Cook, et al., Bioorg Med Chem Lett., 19:773-7 (2009)), Compound 51 (Boehringer Ingelheim from Cook, et al., Bioorg Med Chem Lett., 19:773-7 (2009)), Compound 10n (Boehringer Ingelheim from Riethe, et al., Bioorg Med Chem Lett., 19:1588-91 (2009)), Compound 10o (Boehringer Ingelheim from Riethe, et al., Bioorg Med Chem Lett., 19:1588-91 (2009)), Compound 7v (Vertex from Charrier, et al., J Med Chem., 54:2341-50 (2011)), Compound 7w (Vertex from Charrier, et al., J Med Chem., 54:2341-50 (2011)), Compound 7x (Vertex from Charrier, et al., J Med Chem., 54:2341-50 (2011)), Compound 7y (Vertex from Charrier, et al., J Med Chem., 54:2341-50 (2011)), Compound 44 (Bayer Schering Pharma from vonBonin, et al., Exp Dermatol., 20:41-7 (2011)), Compound 13 (Nycomed from Velankar, et al., Bioorg Med Chem., 18:4547-59 (2010)), Compound 24 (Nycomed from Velankar, et al., Bioorg Med Chem., 18:4547-59 (2010)), Compound 34 (Nycomed from Velankar, et al., Bioorg Med Chem., 18:4547-59 (2010)), Compound 100 (Nycomed from Herdemann, et al., Bioorg Med Chem Lett., 21:1852-6 (2011)), Compound 3 (Sanofi US from McLean, et al., Bioorg Med Chem Lett., 22:3296-300 (2012)), Compound 7 (Sanofi US from McLean, et al., Bioorg Med Chem Lett., 22:3296-300 (2012), and / or or other kinase inhibitors, tyrosine kinase inhibitors, or serine / threonine kinase inhibitors known in the art, as well as any combinations thereof.In any of the foregoing embodiments, pre-treatment regimens comprising ibrutinib (commercially available as IMBRUVICA, and which has the chemical name 1-[(3R)-3-[4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]-1-piperidinyl]-2-propen-1-one) may include orally administering one 140 mg capsule q.d., orally administering two 140 mg capsules q.d., orally administering three 140 mg capsules q.d., or orally administering four 140 mg capsules q.d., for a duration of about one day, two days, three days, four days, five days, six days, seven days, eight days, nine days, ten days, eleven days, twelve days, two weeks, three weeks, one month, two months, three months, four months, five months, or six months. In the foregoing embodiments, pre-treatment regimens comprising ibrutinib may also comprise orally administering an ibrutinib dose selected from the group consisting of 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 375 mg, 400 mg, 425 mg, 450 mg, and 500 mg, wherein the administering occurs once daily, twice daily, three times daily, or four times daily, and wherein the duration of administration is selected from the group consisting of about one day, two days, three days, four days, five days, six days, seven days, eight days, nine days, ten days, eleven days, twelve days, two weeks, three weeks, one month, two months, three months, four months, five months, and six months.

[0304] In some embodiments, the pre-treatment regimen is completed prior to administering a therapeutically effective amount of the pharmaceutical composition disclosed herein. In some embodiments, the pre-treatment regimen may be completed 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 weeks, 49 weeks, 50 weeks, 51 weeks, 52 weeks, or any period of time between any two of these periods prior to administering the therapeutically effective amount of the pharmaceutical composition disclosed herein.

[0305] In any of the foregoing embodiments, the cancer to be treated is a hematological malignancy selected from the group consisting of acute myeloid leukemia (AML), AML without genetic markers or without molecular targets, MDS without genetic markers or without molecular targets, low grade MDS, hypoplastic MDS, CD123 negative (IL-3R negative) AML, FLT3 mutation negative AML, FLT3 positive AML, Nucleophosmin (NPM1) mutation negative acute myeloid leukemia, NPM1 positive AML, mantle cell lymphoma (MCL), follicular lymphoma (FL), diffuse large B cell lymphoma (DLBCL), activated B cell (ABC) DLBCL, germinal center B cell (GCB) DLBCL, chronic lymphocytic leukemia (CLL), CLL with Richter's transformation (or Richter's syndrome), CLL replased after treatment with BTK inhibitors, CLL relapsed after treatment with BCL2 inhibitors, CLL replased after treatment with BTK and BCL2 inhibitors, Relapsed CLL with 17p deletion, small lymphocytic leukemia (SLL), SLL replased after treatment with BTK inhibitors, SLL relapsed after treatment with BCL2 inhibitors, SLL replased after treatment with BTK and BCL2 inhibitors, Relapsed SLL with 17p deletion, CD19 negative B cell leukemia or lymphoma (including CLL / SLL), CD20 negative B cell leukemia or lymphoma (including CLL / SLL), CD23 negative B cell leukemia or lymphoma (including CLL / SLL), non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, relapsed and / or refractory Hodgkin's lymphoma, B cell acute lymphoblastic leukemia (B-ALL), mature B-ALL, Burkitt's lymphoma, Waldenström's macroglobulinemia (WM), multiple myeloma, myelodysplastic syndromes, myelofibrosis, chronic myelocytic leukemia, follicle center lymphoma, indolent NHL, human immunodeficiency virus (HIV) associated B cell lymphoma, Epstein-Barr virus (EBV) associated B cell lymphoma, EBV related lymphoproliferative disorders and post-transplant lymphoproliferative disorders.

[0306] The invention provides any of the foregoing embodiments modified as applicable such that the cancer to be treated is either resistant or refractory to treatment with an ITK inhibitor, such as ibrutinib, or has relapsed following a response to treatment with an ITK inhibitor, such as ibrutinib. The invention provides any of the foregoing embodiments modified as applicable such that the cancer to be treated is either resistant or refractory to treatment with a BTK inhibitor, such as described elsewhere herein, or has relapsed following a response to treatment with a BTK inhibitor, such as described elsewhere herein. The invention provides any of the foregoing embodiments modified as applicable such that the cancer to be treated is either resistant or refractory to treatment with a BCL2 inhibitor, such as described elsewhere herein, or has relapsed following a response to treatment with a BCL2 inhibitor, such as described elsewhere herein.

[0307] In any of the foregoing embodiments of the invention, pre-treatment with BTK inhibitor is described. Bruton's tyrosine kinase (BTK) also known as tyrosine-protein kinase BTK, is a tyrosine kinase that is encoded by the BTK gene in humans. BTK plays a crucial role in B cell development. Any BTK inhibitor known in the art may be used in embodiments of the present invention. See, for example, Tasso, et al. in “The Development of BTK Inhibitors: A Five-Year Update” Molecules. 2021 Dec. 6; 26(23):7411, doi: 10.3390 / molecules26237411, which is incorporated herein by reference in its entirety.

[0308] In an embodiment of the present invention, the BTK inhibitor is selected from the group consisting of 1,3,5-Triazine-based BTK inhibitors, Diphenylaminopyrimidines-based BTK inhibitors, Pyridinone-based BTK inhibitors, Pyrazolo-Pyrimidine-based BTK inhibitors, Tieno-Pyrimidine-based BTK inhibitors, Pyrrolo[2,3-d]pyrimidine-based BTK inhibitors, Imidazo-Pyrazine-based BTK inhibitors, Imidazo-Pyridine-based BTK inhibitors, Imidazo-Pyrazole-based BTK inhibitors, Quinoline-based BTK inhibitors, Isoquinoline-based BTK inhibitors, Phthalazine-based BTK inhibitors, Carbazole-based BTK inhibitors, Tetrahydrocarbazole-based BTK inhibitors, Indole-based BTK inhibitors, pyrano-chromenones-based BTK inhibitors, benzofuro-pyridines-based BTK inhibitors, pyrazoles-based BTK inhibitors, thiazoles-based BTK inhibitors, proteolysis-targeting chimera compound-based BTK inhibitors, and any other BTK inhibitors known in the art. In an embodiment of the invention, pre-treatment regimens with a BTK inhibitor are as known in the art and / or prescribed by a physician. In an embodiment of the invention, the BTK inhibitor is selected from the group consisting of ibrutinib or another BTK inhibitor, such as irreversible BTK inhibitors such as acalabrutinib, zanubrutinib, tirabrutinib, orelabrutinib, evobrutinib, spebrutinib, remibrutinib, tolebrutinib, olmutinib, branebrutinib, TAK-020, elsubrutinib, rilzabrutinib, and reversible BTK inhibitors such as, vecabrutinib, GNE-431, RN-486, BMS935177, BMS-986142, CGI-1746, GDC-0834, G-744, G-278, and fenebrutinib and combinations thereof. In an embodiment of the invention, the BTK inhibitor is selected from the group consisting of:and combinations thereof.In any of the foregoing embodiments, pre-treatment regimens comprising ibrutinib (commercially available as IMBRUVICA, and which has the chemical name 1-[(3R)-3-[4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]-1-piperidinyl]-2-propen-1-one) may include orally administering one 140 mg capsule q.d., orally administering two 140 mg capsules q.d., orally administering three 140 mg capsules q.d., or orally administering four 140 mg capsules q.d., for a duration of about one day, two days, three days, four days, five days, six days, seven days, eight days, nine days, ten days, eleven days, twelve days, two weeks, three weeks, one month, two months, three months, four months, five months, or six months. In the foregoing embodiments, pre-treatment regimens comprising ibrutinib may also comprise orally administering an ibrutinib dose selected from the group consisting of 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 375 mg, 400 mg, 425 mg, 450 mg, and 500 mg, wherein the administering occurs once daily, twice daily, three times daily, or four times daily, and wherein the duration of administration is selected from the group consisting of about one day, two days, three days, four days, five days, six days, seven days, eight days, nine days, ten days, eleven days, twelve days, two weeks, three weeks, one month, two months, three months, four months, five months, and six months.

[0310] In some embodiments, the pre-treatment regimen is completed prior to administering a therapeutically effective amount of the pharmaceutical composition disclosed herein. In some embodiments, the pre-treatment regimen may be completed 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 weeks, 49 weeks, 50 weeks, 51 weeks, 52 weeks, or any period of time between any two of these periods prior to administering the therapeutically effective amount of the pharmaceutical composition disclosed herein.

[0311] In any of the foregoing embodiments, the cancer to be treated is a hematological malignancy selected from the group consisting of acute myeloid leukemia (AML), AML without genetic markers or without molecular targets, MDS without genetic markers or without molecular targets, low grade MDS, hypoplastic MDS, CD123 negative (IL-3R negative) AML, FLT3 mutation negative AML, FLT3 positive AML, Nucleophosmin (NPM1) mutation negative acute myeloid leukemia, NPM1 positive AML, mantle cell lymphoma (MCL), follicular lymphoma (FL), diffuse large B cell lymphoma (DLBCL), activated B cell (ABC) DLBCL, germinal center B cell (GCB) DLBCL, chronic lymphocytic leukemia (CLL), CLL with Richter's transformation (or Richter's syndrome), CLL replased after treatment with BTK inhibitors, CLL relapsed after treatment with BCL2 inhibitors, CLL replased after treatment with BTK and BCL2 inhibitors, Relapsed CLL with 17p deletion, small lymphocytic leukemia (SLL), SLL replased after treatment with BTK inhibitors, SLL relapsed after treatment with BCL2 inhibitors, SLL replased after treatment with BTK and BCL2 inhibitors, Relapsed SLL with 17p deletion, CD19 negative B cell leukemia or lymphoma (including CLL / SLL), CD20 negative B cell leukemia or lymphoma (including CLL / SLL), CD23 negative B cell leukemia or lymphoma (including CLL / SLL), non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, relapsed and / or refractory Hodgkin's lymphoma, B cell acute lymphoblastic leukemia (B-ALL), mature B-ALL, Burkitt's lymphoma, Waldenström's macroglobulinemia (WM), multiple myeloma, myelodysplastic syndromes, myelofibrosis, chronic myelocytic leukemia, follicle center lymphoma, indolent NHL, human immunodeficiency virus (HIV) associated B cell lymphoma, Epstein-Barr virus (EBV) associated B cell lymphoma, EBV related lymphoproliferative disorders and post-transplant lymphoproliferative disorders.

[0312] In any of the foregoing embodiments of the invention, pre-treatment with a BCL2 inhibitor is described. The BCL2 proteins are oncogenes that promote cell survival and are frequently upregulated in multiple myeloma. Any BCL2 inhibitors known in the art may be used in embodiments of the present invention. See, e.g., Gupta et al. in “BCL2 Family Inhibitors in the Biology and Treatment of Multiple Myeloma,” Blood and Lymphatic Cancer: Targets and Therapy 2021:11 11-24, which is incorporated herein by reference in its entirety. In some embodiments, the BCL2 inhibitor is selected from the group consisting of a BH3 mimetic BCL2 inhibitor, venetoclax, obatoclax, subatoclax, maritoclax, gossypol, apogossypol, TW-37, UMI-77, BDA-366, navitoclax, ABT-737, any other BCL2 inhibitor known in the art, and combinations thereof. In an embodiment of the invention, the BCL2 inhibitor is selected from the group consisting:and combinations thereof. In any of the foregoing embodiments, pre-treatment regimens comprising venteoclax (commercially available as Venclexta) may include orally administering one 100 mg capsule q.d., orally administering two 100 mg capsules q.d., orally administering three 100 mg capsules q.d., or orally administering four 100 mg capsules q.d., for a duration of about one day, two days, three days, four days, five days, six days, seven days, eight days, nine days, ten days, eleven days, twelve days, two weeks, three weeks, one month, two months, three months, four months, five months, or six months. In the foregoing embodiments, pre-treatment regimens comprising venetoclax may also comprise orally administering a venetoclax dose selected from the group consisting of 20 mg, 40 mg, 60 mg, 80 mg, 100 mg, 120 mg, 140 mg, 160 mg, 180 mg, 200 mg, 220 mg, 240 mg, 260 mg, 280 mg, 300 mg, 320 mg, 340 mg, 360 mg, 380 mg, 400 mg, 420 mg, 440 mg, 460 mg, 480 mg, and 500 mg, wherein the administering occurs once daily, twice daily, three times daily, or four times daily, and wherein the duration of administration is selected from the group consisting of about one day, two days, three days, four days, five days, six days, seven days, eight days, nine days, ten days, eleven days, twelve days, two weeks, three weeks, one month, two months, three months, four months, five months, and six months.In some embodiments, the pre-treatment regimen is completed prior to administering a therapeutically effective amount of the pharmaceutical composition disclosed herein. In some embodiments, the pre-treatment regimen may be completed 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 weeks, 49 weeks, 50 weeks, 51 weeks, 52 weeks, or any period of time between any two of these periods prior to administering the therapeutically effective amount of the pharmaceutical composition disclosed herein.

[0314] In any of the foregoing embodiments, the cancer to be treated is a hematological malignancy selected from the group consisting of acute myeloid leukemia (AML), AML without genetic markers or without molecular targets, MDS without genetic markers or without molecular targets, low grade MDS, hypoplastic MDS, CD123 negative (IL-3R negative) AML, FLT3 mutation negative AML, FLT3 positive AML, Nucleophosmin (NPM1) mutation negative acute myeloid leukemia, NPM1 positive AML, mantle cell lymphoma (MCL), follicular lymphoma (FL), diffuse large B cell lymphoma (DLBCL), activated B cell (ABC) DLBCL, germinal center B cell (GCB) DLBCL, chronic lymphocytic leukemia (CLL), CLL with Richter's transformation (or Richter's syndrome), CLL replased after treatment with BTK inhibitors, CLL relapsed after treatment with BCL2 inhibitors, CLL replased after treatment with BTK and BCL2 inhibitors, Relapsed CLL with 17p deletion, small lymphocytic leukemia (SLL), SLL replased after treatment with BTK inhibitors, SLL relapsed after treatment with BCL2 inhibitors, SLL replased after treatment with BTK and BCL2 inhibitors, Relapsed SLL with 17p deletion, CD19 negative B cell leukemia or lymphoma (including CLL / SLL), CD20 negative B cell leukemia or lymphoma (including CLL / SLL), CD23 negative B cell leukemia or lymphoma (including CLL / SLL), non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, relapsed and / or refractory Hodgkin's lymphoma, B cell acute lymphoblastic leukemia (B-ALL), mature B-ALL, Burkitt's lymphoma, Waldenström's macroglobulinemia (WM), multiple myeloma, myelodysplastic syndromes, myelofibrosis, chronic myelocytic leukemia, follicle center lymphoma, indolent NHL, human immunodeficiency virus (HIV) associated B cell lymphoma, Epstein-Barr virus (EBV) associated B cell lymphoma, EBV related lymphoproliferative disorders and post-transplant lymphoproliferative disorders.

[0315] Efficacy of the methods and compositions described herein in treating, preventing and / or managing the indicated diseases or disorders can be tested using various animal models known in the art.Non-Myeloablative Lymphodepletion with Chemotherapy

[0316] In an embodiment, the invention provides a method of treating a cancer with a population of TILs, wherein a patient is pre-treated with non-myeloablative chemotherapy prior to an infusion of TILs according to the present disclosure.

[0317] In an embodiment, the non-myeloablative chemotherapy is one or more chemotherapeutic agents. In an embodiment, the non-myeloablative chemotherapy is cyclophosphamide 60 mg / kg / d for 2 days (days 27 and 26 prior to TIL infusion) and fludarabine 25 mg / m2 / d for 5 days (days 27 to 23 prior to TIL infusion). In an embodiment, after non-myeloablative chemotherapy and TIL infusion (at day 0) according to the present disclosure, the patient receives an intravenous infusion of IL-2 intravenously at 720,000 IU / kg every 8 hours to physiologic tolerance. Experimental findings indicate that lymphodepletion prior to adoptive transfer of tumor-specific T lymphocytes plays a key role in enhancing treatment efficacy by eliminating regulatory T cells and competing elements of the immune system (“cytokine sinks”). Accordingly, some embodiments of the invention utilize a lymphodepletion step (sometimes also referred to as “immunosuppressive conditioning”) on the patient prior to the introduction of the TILs of the invention.

[0318] In general, lymphodepletion is achieved using administration of fludarabine or cyclophosphamide (the active form being referred to as mafosfamide) and combinations thereof. Such methods are described in Gassner, et al., Cancer Immunol. Immunother. 2011, 60, 75-85, Muranski, et al., Nat. Clin. Pract. Oncol., 2006, 3, 668-681, Dudley, et al., J. Clin. Oncol. 2008, 26, 5233-5239, and Dudley, et al., J. Clin. Oncol. 2005, 23, 2346-2357, all of which are incorporated by reference herein in their entireties.

[0319] In some embodiments, the fludarabine is administered at a concentration of 0.5 μg / mL-10 μg / mL fludarabine. In some embodiments, the fludarabine is administered at a concentration of 1 μg / mL fludarabine. In some embodiments, the fludarabine treatment is administered for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days or more. In some embodiments, the fludarabine is administered at a dosage of 10 mg / kg / day, 15 mg / kg / day, 20 mg / kg / day, 25 mg / kg / day, 30 mg / kg / day, 35 mg / kg / day, 40 mg / kg / day, or 45 mg / kg / day. In some embodiments, the fludarabine treatment is administered for 2-7 days at 35 mg / kg / day. In some embodiments, the fludarabine treatment is administered for 4-5 days at 35 mg / kg / day. In some embodiments, the fludarabine treatment is administered for 4-5 days at 25 mg / kg / day.

[0320] In some embodiments, the mafosfamide, the active form of cyclophosphamide, is obtained at a concentration of 0.5 μg / mL-10 μg / mL by administration of cyclophosphamide. In some embodiments, mafosfamide, the active form of cyclophosphamide, is obtained at a concentration of 1 μg / mL by administration of cyclophosphamide. In some embodiments, the cyclophosphamide treatment is administered for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days or more. In some embodiments, the cyclophosphamide is administered at a dosage of 100 mg / m2 / day, 150 mg / m2 / day, 175 mg / m2 / day, 200 mg / m2 / day, 225 mg / m2 / day, 250 mg / m2 / day, 275 mg / m2 / day, or 300 mg / m2 / day. In some embodiments, the cyclophosphamide is administered intravenously (i.e., i.v.) In some embodiments, the cyclophosphamide treatment is administered for 2-7 days at 35 mg / kg / day. In some embodiments, the cyclophosphamide treatment is administered for 4-5 days at 250 mg / m2 / day i.v. In some embodiments, the cyclophosphamide treatment is administered for 4 days at 250 mg / m2 / day i.v.

[0321] In some embodiments, lymphodepletion is performed by administering the fludarabine and the cyclophosphamide are together to a patient. In some embodiments, fludarabine is administered at 25 mg / m2 / day iv. and cyclophosphamide is administered at 250 mg / m2 / day i.v. over 4 days.

[0322] In an embodiment, the lymphodepletion is performed by administration of cyclophosphamide at a dose of 60 mg / m2 / day for two days followed by administration of fludarabine at a dose of 25 mg / m2 / day for five days.

[0323] In an embodiment, the non-myeloablative preparative regimen, consisting of daily cyclophosphamide 500 mg / m2 / day and fludarabine 30 mg / m2 / day administered by intravenous (IV) over 30 minutes on Day −5, Day −4, and Day −3. Day −2 and Day −1 are rest days.

[0324] Several methods of expanding TILs obtained from bone marrow or peripheral blood are described herein. In an embodiment of the invention, the lymphodepletion is performed by administration of cyclophosphamide at a dose of 60 mg / m2 / day for two days followed by administration of fludarabine at a dose of 25 mg / m2 / day for five days. Several methods of expanding TILs obtained from bone marrow or peripheral blood are described herein.Methods of Expanding Peripheral Blood Lymphocytes (PBLs) from Peripheral Blood

[0325] In an embodiment of the invention, PBLs are expanded using the processes described herein. In an embodiment of the invention, the method comprises obtaining a PBMC sample from whole blood. In an embodiment, the method comprises enriching T-cells by isolating pure T-cells from PBMCs using positive selection of a CD3+ / CD28+ fraction, as follows. Thaw the cryopreserved PBMCs in a 37° C. waterbath. Transfer the thawed PBMCs into a 50 mL conical tube and mix well. Divide the cell suspension into two equal portions into two appropriately labelled 15 mL polystyrene conical tubes. Pellet the cells in the 15 mL tubes via centrifugation 400 g for 5 minutes at 24° C. (acceleration=9, deceleration=9). During centrifugation, mix the CTS Dynabeads (CD3 / CD28) by placing on a rocker for at least 5 minutes. Remove the cells from the centrifuge and aspirate all the media. Cap tubes and scrape them along a rough surface (such as a tube rack) to help break up cell pellet. Calculate and record the number of CD3+ viable cells in a tube labelled appropriately (for example, “Method #1: Number of CD3+ viable cells=% CD3+cells*TVC” (total viable cells). Resuspend the cells in a tube labelled appropriately (for example, “Method 1”) so that the concentration of the viable T-cells is le7 / mL using wash buffer (sterile phosphate buffered saline (PBS), 1% Human Serum Albumin, 10 U / mL Dnase). Add the washed CTS DynaBeads (CD3 / 28) at 3 beads: 1 T-cell ratio by transferring the volume as calculated above. Incubate the sample with the Dynabeads, in a microtube covered with foil, on a rocker (1-3 RPM end to end) at room temperature for 30 minutes in the dark. After 30 minutes of incubation, place the sample in a 15 mL conical tube, rinse the microtube with 1 mL of CM2+IL-2 (3000 IU / mL) and transfer to the 15 mL tube. Bring the volume up to 10 mL using CM2+IL-2 and mix well using a pipettor. Place the tube on the DynaMag-15 for one to two minutes for positive selection of the bead-bound CD3+ cells. Decant the cell suspension (negative portion) into a 50 mL conical tube labelled appropriately (for example, “Method #1—no T cell fraction”). Immediately add 10 mL of CM2 media with IL-2 (3000 IU / mL) to the 15 mL tube that contains the bead-bound cells and mix. Place the tube on the Dynamag-15 for one to two minutes. Decant the cell suspension (residual negative portion) into the 50 mL conical tube labeled appropriately (for example, “Method #1—no T cell fraction”). Immediately add 5 mL of CM2 media with IL-2 (3000 IU / mL) to the 15 mL tube that contains the bead-bound cells and mix. Relabel the tube appropriately (for example, “Method #1—T cell fraction”). Count negative and positive portions. Obtain about 5e5 cells from each of the negative and the positive portions for flow analysis (CD3 / 4 / 8 / 19 / 14) of the fresh sample. CD3+CD8+ cells are CTLs, CD3+CD4+ cells are helper T-cells, CD19 cells are B-cells, and CD14+ cells are macrophages. Cryopreserve the leftover negative portion. Proceed with the culture of the positive T-cell enriched portion along with the Dynabeads.

[0326] On Day 0, to each of two G-REX5M flasks, place le6 viable T-cells. Label the flasks appropriately (for example, “Method #1”). Alternatively, to each G-REX 10M, place a minimum of 2e6 viable T cells. Slowly bring up the volume of the media in each G-REX5M flask to 20 mL of CM2 supplemented with 30001U IL-2 / mL or to 40 mL in each G-REX10M. Place the flasks in the incubator (37° C. 5% CO2).

[0327] On Day 4, add media. If cultured in G-REX 5M, add 20 mL of CM4+IL-2 (3000 IU / mL). If cultured in G-REX 10M, add 40 mL of CM4+IL-2 (3000 IU / mL).

[0328] On Day 7, add media. If cultured in G-REX 5M, add 10 mL of CM4+IL-2 (3000 IU / mL). If cultured in G-REX 10M, add 20 mL of CM4+IL-2 (3000 IU / mL).

[0329] Cells may be harvested on Day 9 or Day 11.

[0330] On the day of harvest, harvest one G-REX flask from each enrichment condition. Reduce the volume in the media to about 10% without disturbing the cells. Save two 1 mL samples for metabolite analysis at −20° C. freezer. Resuspend the cells and harvest in a 50 mL conical labelled appropriately (for example, “Method #1”). Add about 10 mL of Plasmalyte +1% HSA to each 50 mL tube. Place the conical tube in a Dynamag-50 for one to two minutes for bead removal. Using a 5 or 10 mL pipette, remove the cell suspension into anther 50 mL conical tube labelled Method #1 final. Immediately add 10 mL of Plasmalyte+1% HSA into the tubes in the Dynamag-50. Remove them from the magnet and mix, then return to the magnet. Place the 50 mL conicals again on the DynaMag-50 for 2 minutes to rinse. Using a 5 or 10 mL pipette, remove the cell suspension into the 50 mL conical tube labelled appropriately (for example, “Method #1 final”). Remove a sample for cell count and viability and for bead residual count. Cryopreserve the final product in vials using chilled freeze media (for example, 49.9% Plasmalyte-A, 0.5% HSA and 50% CS10).

[0331] In an embodiment, the invention provides a method for expanding peripheral blood lymphocytes (PBLs) from peripheral blood comprising:

[0332] b. Obtaining a sample of peripheral blood mononuclear cells (PBMCs) from the peripheral blood of a patient, wherein said sample is optionally cryopreserved and the patient is optionally pretreated with an ITK inhibitor;

[0333] c. Optionally washing the PBMCs by centrifugation;

[0334] d. Admixing magnetic beads selective for CD3 and CD28 to the PBMCs to form an admixture of the beads and the PBMCs;

[0335] e. Seeding the admixture of the beads and the PBMCs into a gas-permeable container and co-culturing said PBMCs in media comprising about 3000 IU / mL of IL-2 in for about 4 to about 6 days;

[0336] f. Feeding said PBMCs using media comprising about 3000 IU / mL of IL-2, and co-culturing said PBMCs for about 5 days, such that the total co-culture period of steps d and e is about 9 to about 11 days;

[0337] g. Harvesting PBMCs from media;

[0338] h. Removing the magnetic beads selective for CD3 and CD28 from the harvested PBMCs by using a magnet;

[0339] i. Removing residual B-cells from the harvested PBMCs using magnetic-activated cell sorting and beads selective for CD19 to provide a PBL product;

[0340] j. Washing and concentrating the PBL product using a cell harvester; and

[0341] k. Formulating and optionally cryopreserving the PBL product, wherein the ITK inhibitor is optionally an ITK inhibitor that covalently binds to ITK.

[0342] In an embodiment, PBMCs are isolated from a whole blood sample. In an embodiment, the PBMC sample is used as the starting material to expand the PBLs. In an embodiment, the PBMC sample is cryopreserved prior to the expansion process. In another embodiment, a fresh PBMC sample is used as the starting material to expand the PBLs. In an embodiment of the invention, T-cells are isolated from PBMCs using methods known in the art. In an embodiment, the T-cells are isolated using a Human Pan T-cell isolation kit and LS columns. In an embodiment of the invention, T-cells are isolated from PBMCs using antibody selection methods known in the art, for example, CD19 negative selection.

[0343] In an embodiment of the invention, the process is performed over about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, or about 14 days. In another embodiment, the process is performed over about 7 days. In another embodiment, the process is performed over about 14 days.

[0344] In an embodiment of the invention, the PBMCs are cultured with antiCD3 / antiCD28 antibodies. In an embodiment, any available antiCD3 / antiCD28 product is useful in the present invention. In an embodiment of the invention, the commercially available product used are DynaBeads*. In an embodiment, the DynaBeads® are cultured with the PBMCs in a ratio of 1:1 (beads:cells). In another embodiment, the antibodies are DynaBeads® cultured with the PBMCs in a ratio of 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, or 5:1 (beads:cells). In an embodiment of the invention, the antibody culturing steps and / or the step of restimulating cells with antibody is performed over a period of from about 2 to about 6 days, from about 3 to about 5 days, or for about 4 days. In an embodiment of the invention, the antibody culturing step is performed over a period of about 2 days, 3 days, 4 days, 5 days, or 6 days.

[0345] In an embodiment, the PBMC sample is cultured with IL-2. In an embodiment of the invention, the cell culture medium used for expansion of the PBLs from PBMCs comprises IL-2 at a concentration selected from the group consisting of about 100 IU / mL, about 200 IU / mL, about 300 IU / mL, about 400 IU / mL, about 100 IU / mL, about 100 IU / mL, about 100 IU / mL, about 100 IU / mL, about 100 IU / mL, about 500 IU / mL, about 600 IU / mL, about 700 IU / mL, about 800 IU / mL, about 900 IU / mL, about 1,000 IU / mL, about 1,100 IU / mL, about 1,200 IU / mL, about 1,300 IU / mL, about 1,400 IU / mL, about 1,500 IU / mL, about 1,600 IU / mL, about 1,700 IU / mL, about 1,800 IU / mL, about 1,900 IU / mL, about 2,000 IU / mL, about 2,100 IU / mL, about 2,200 IU / mL, about 2,300 IU / mL, about 2,400 IU / mL, about 2,500 IU / mL, about 2,600 IU / mL, about 2,700 IU / mL, about 2,800 IU / mL, about 2,900 IU / mL, about 3,000 IU / mL, about 3,100 IU / mL, about 3,200 IU / mL, about 3,300 IU / mL, about 3,400 IU / mL, about 3,500 IU / mL, about 3,600 IU / mL, about 3,700 IU / mL, about 3,800 IU / mL, about 3,900 IU / mL, about 4,000 IU / mL, about 4,100 IU / mL, about 4,200 IU / mL, about 4,300 IU / mL, about 4,400 IU / mL, about 4,500 IU / mL, about 4,600 IU / mL, about 4,700 IU / mL, about 4,800 IU / mL, about 4,900 IU / mL, about 5,000 IU / mL, about 5,100 IU / mL, about 5,200 IU / mL, about 5,300 IU / mL, about 5,400 IU / mL, about 5,500 IU / mL, about 5,600 IU / mL, about 5,700 IU / mL, about 5,800 IU / mL, about 5,900 IU / mL, about 6,000 IU / mL, about 6,500 IU / mL, about 7,000 IU / mL, about 7,500 IU / mL, about 8,000 IU / mL, about 8,500 IU / mL, about 9,000 IU / mL, about 9,500 IU / mL, and about 10,000 IU / mL.

[0346] In an embodiment of the invention, the starting cell number of PBMCs for the expansion process is from about 25,000 to about 1,000,000, from about 30,000 to about 900,000, from about 35,000 to about 850,000, from about 40,000 to about 800,000, from about 45,000 to about 800,000, from about 50,000 to about 750,000, from about 55,000 to about 700,000, from about 60,000 to about 650,000, from about 65,000 to about 600,000, from about 70,000 to about 550,000, preferably from about 75,000 to about 500,000, from about 80,000 to about 450,000, from about 85,000 to about 400,000, from about 90,000 to about 350,000, from about 95,000 to about 300,000, from about 100,000 to about 250,000, from about 105,000 to about 200,000, or from about 110,000 to about 150,000. In an embodiment of the invention, the starting cell number of PBMCs is about 138,000, 140,000, 145,000, or more. In another embodiment, the starting cell number of PBMCs is about 28,000. In another embodiment, the starting cell number of PBMCs is about 62,000. In another embodiment, the starting cell number of PBMCs is about 338,000. In another embodiment, the starting cell number of PBMCs is about 336,000. In another embodiment, the starting cell number of PBMCs is 1 million, 2 million, 3 million, 4 million, 5 million, 6 million, 7 million, 8 million, 9 million, 10 million or more. In another embodiment, the starting cell number of PBMCs is 1 million to 10 million, 2 million to 9 million, 3 million to 8 million, 4 million to 7 million, or 5 million to 6 million. In another embodiment, the starting cell number of PBMCs is about 4 million. In yet another embodiment, the starting cell number of PBMCs is at least about 4 million, at least about 5 million, or at least about 6 million or more.

[0347] In an embodiment of the invention, the cells are grown in a GRex 24 well plate. In an embodiment of the invention, a comparable well plate is used. In an embodiment, the starting material for the expansion is about 5×105 T-cells per well. In an embodiment of the invention, there are 1×106 cells per well. In an embodiment of the invention, the number of cells per well is sufficient to seed the well and expand the T-cells.

[0348] In an embodiment of the invention, the cells are grown in a GRex 100MCS container. In an embodiment of the invention, a comparable container is used. In an embodiment, the starting material for expansion is seeded at a density of about 25,000 to about 50,000 T-cells per square centimeter.

[0349] In an embodiment of the invention, the fold expansion of PBLs is from about 20% to about 100%, 25% to about 95%, 30% to about 90%, 35% to about 85%, 40% to about 80%, 45% to about 75%, 50% to about 100%, or 25% to about 75%. In an embodiment of the invention, the fold expansion is about 25%. In another embodiment of the invention, the fold expansion is about 50%. In another embodiment, the fold expansion is about 75%.

[0350] In an embodiment of the invention, additional IL-2 may be added to the culture on one or more days throughout the process. In an embodiment of the invention, additional IL-2 is added on Day 4. In an embodiment of the invention, additional IL-2 is added on Day 7. In an embodiment of the invention, additional IL-2 is added on Day 11. In another embodiment, additional IL-2 is added on Day 4, Day 7, and / or Day 11. In an embodiment of the invention, the cell culture medium may be changed on one or more days through the cell culture process. In an embodiment, the cell culture medium is changed on Day 4, Day 7, and / or Day 11 of the process. In an embodiment of the invention, the PBLs are cultured with additional IL-2 for a period of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days. In an embodiment of the invention, PBLs are cultured for a period of 3 days after each addition of IL-2.

[0351] In some embodiments, the first expansion culture medium is referred to as “CM”, an abbreviation for culture media. In some embodiments, it is referred to as CM1 (culture medium 1). In some embodiments, CM consists of RPMI 1640 with GlutaMAX, supplemented with 10% human AB serum, 25 mM Hepes, and 10 mg / mL gentamicin. In some embodiments, the CM is the CM1 described in the Examples. In some embodiments, the first expansion occurs in an initial cell culture medium or a first cell culture medium.

[0352] In an embodiment, the cell culture medium is exchanged at least one time during the method. In an embodiment, the cell culture medium is exchanged at the same time that additional IL-2 is added. In another embodiment the cell culture medium is exchanged on at least one of Day 1, Day 2, Day 3, Day 4, Day 5, Day 6, Day 7, Day 8, Day 9, Day 10, Day 11, Day 12, Day 13, or Day 14. In an embodiment of the invention, the cell culture medium used throughout the method may be the same or different. In an embodiment of the invention, the cell culture medium is CM-2, CM-4, or AIM-V.

[0353] In an embodiment of the invention, T-cells may be restimulated with antiCD3 / antiCD28 antibodies on one or more days throughout the 14-day expansion process. In an embodiment, the T-cells are restimulated on Day 7. In an embodiment, GRex 10M flasks are used for the restimulation step. In an embodiment of the invention, comparable flasks are used.

[0354] In an embodiment of the invention, the DynaBeads® are removed using a DynaMag™ Magnet, the cells are counted, and the cells are analyzed using phenotypic and functional analysis as further described in the Examples below. In an embodiment of the invention, antibodies are separated from the PBLs or MILs using methods known in the art. In any of the foregoing embodiments, magnetic bead-based selection of TILs, PBLs, or MILs is used.

[0355] In an embodiment of the invention, the PBMC sample is incubated for a period of time at a desired temperature effective to identify the non-adherent cells. In an embodiment of the invention, the incubation time is about 3 hours. In an embodiment of the invention, the temperature is about 370 Celsius. The non-adherent cells are then expanded using the process described above.

[0356] In an embodiment of the invention, the PBMCs are obtained from a patient who has been treated with ibrutinib or another ITK or kinase inhibitor, such ITK and kinase inhibitors as described elsewhere herein. In an embodiment of the invention, the ITK inhibitor is a covalent ITK inhibitor that covalently and irreversibly binds to ITK. In an embodiment of the invention, the ITK inhibitor is an allosteric ITK inhibitor that binds to ITK. In an embodiment of the invention, the PBMCs are obtained from a patient who has been treated with ibrutinib or other ITK inhibitor, including ITK inhibitors as described elsewhere herein, prior to obtaining a PBMC sample for use with any of the foregoing methods, including PBL Method 1. In an embodiment of the invention, the ITK inhibitor treatment has been administered at least 1 time, at least 2, times, or at least 3 times or more. In an embodiment of the invention, PBLs that are expanded from patients pretreated with ibrutinib or other ITK inhibitor comprise less LAG3+, PD-1+ cells than those expanded from patients not pretreated with ibrutinib or other ITK inhibitor. In an embodiment of the invention PBLs that are expanded from patients pretreated with ibrutinib or other ITK inhibitor comprise increased levels of IFNγ production than those expanded from patients not pretreated with ibrutinib or other ITK inhibitor. In an embodiment of the invention, PBLs that are expanded from patients pretreated with ibrutinib or other ITK inhibitor comprise increased lytic activity at lower Effector:Target cell ratios than those expanded from patients not pretreated with ibrutinib or other ITK inhibitor. In an embodiment of the invention, patients pretreated with ibrutinib or other ITK inhibitor have higher fold-expansion as compared with untreated patients.

[0357] In an embodiment of the invention, the method includes a step of adding an ITK inhibitor to the cell culture. In an embodiment, the ITK inhibitor is added on one or more of Day 0, Day 1, Day 2, Day 3, Day 4, Day 5, Day 6, Day 7, Day 8, Day 9, Day 10, Day 11, Day 12, Day 13, or Day 14 of the process. In an embodiment, the ITK inhibitor is added on the days during the method when cell culture medium is exchanged. In an embodiment, the ITK inhibitor is added on Day 0 and when cell culture medium is exchanged. In an embodiment, the ITK inhibitor is added during the method when IL-2 is added. In an embodiment, the ITK inhibitor is added on Day 0, Day 4, Day 7, and optionally Day 11 of the method. In an embodiment of the invention, the ITK inhibitor is added at Day 0 and at Day 7 of the method. In an embodiment of the invention, the ITK inhibitor is one known in the art. In an embodiment of the invention, the ITK inhibitor is one described elsewhere herein.

[0358] In an embodiment of the invention, the ITK inhibitor is used in the method at a concentration of from about 0.1 nM to about 5 uM. In an embodiment, the ITK inhibitor is used in the method at a concentration of about 0.1 nM, 0.5 nM, 1 nM, 5 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, 400 nM, 450 nM, 500 nM, 550 nM, 600 nM, 650 nM, 700 nM, 750 nM, 800 nM, 850 nM, 900 nM, 950 nM, 1 uM, 2 uM, 3 uM, 4 uM, or 5 uM.

[0359] In an embodiment of the invention, the method includes a step of adding an ITK inhibitor when the PBMCs are derived from a patient who has no prior exposure to an ITK inhibitor treatment, such as ibrutinib.

[0360] In some embodiments, the PBMC sample is from a subject or patient who has been optionally pre-treated with a regimen comprising a kinase inhibitor or an ITK inhibitor. In some embodiments, the tumor sample is from a subject or patient who has been pre-treated with a regimen comprising a kinase inhibitor or an ITK inhibitor. In some embodiments, the PBMC sample is from a subject or patient who has been pre-treated with a regimen comprising a kinase inhibitor or an ITK inhibitor, has undergone treatment for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, or 1 year or more. In another embodiment, the PBMCs are derived from a patient who is currently on an ITK inhibitor regimen, such as ibrutinib.

[0361] In some embodiments, the PBMC sample is from a subject or patient who has been pre-treated with a regimen comprising a kinase inhibitor or an ITK inhibitor and is refractory to treatment with a kinase inhibitor or an ITK inhibitor, such as ibrutinib.

[0362] In some embodiments, the PBMC sample is from a subject or patient who has been pre-treated with a regimen comprising a kinase inhibitor or an ITK inhibitor but is no longer undergoing treatment with a kinase inhibitor or an ITK inhibitor. In some embodiments, the PBMC sample is from a subject or patient who has been pre-treated with a regimen comprising a kinase inhibitor or an ITK inhibitor but is no longer undergoing treatment with a kinase inhibitor or an ITK inhibitor and has not undergone treatment for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, or at least 1 year or more. In another embodiment, the PBMCs are derived from a patient who has prior exposure to an ITK inhibitor, but has not been treated in at least 3 months, at least 6 months, at least 9 months, or at least 1 year.

[0363] In an embodiment of the invention, the PBMCs are obtained from a patient who has been treated with ibrutinib or another BTK inhibitor, such as irreversible BTK inhibitors such as acalabrutinib, zanubrutinib, tirabrutinib, orelabrutinib, evobrutinib, spebrutinib, remibrutinib, tolebrutinib, olmutinib, branebrutinib, TAK-020, elsubrutinib, rilzabrutinib, and reversible BTK inhibitors such as, vecabrutinib, GNE-431, RN-486, BMS935177, BMS-986142, CGI-1746, GDC-0834, G-744, G-278, and fenebrutinib. Other BTK inhibitors may include those described by Tasso, et al. in “The Development of BTK Inhibitors: A Five-Year Update” Molecules. 2021 Dec. 6; 26(23):7411, doi: 10.3390 / molecules26237411, which is incorporated herein by reference in its entirety.

[0364] In an embodiment of the invention, the BTK inhibitor is a covalent BTK inhibitor that covalently and irreversibly binds to BTK. In an embodiment of the invention, the PBMCs are obtained from a patient who has been treated with ibrutinib or other BTK inhibitor, including BTK inhibitors as described elsewhere herein, prior to obtaining a PBMC sample for use with any of the foregoing methods, including PBL Method 1. In an embodiment of the invention, the BTK inhibitor treatment has been administered at least 1 time, at least 2, times, or at least 3 times or more. In an embodiment of the invention, PBLs that are expanded from patients pretreated with ibrutinib or other BTK inhibitor comprise less LAG3+, PD-1+ cells than those expanded from patients not pretreated with ibrutinib or other BTK inhibitor. In an embodiment of the invention PBLs that are expanded from patients pretreated with ibrutinib or other BTK inhibitor comprise increased levels of IFNγ production than those expanded from patients not pretreated with ibrutinib or other BTK inhibitor. In an embodiment of the invention, PBLs that are expanded from patients pretreated with ibrutinib or other BTK inhibitor comprise increased lytic activity at lower Effector:Target cell ratios than those expanded from patients not pretreated with ibrutinib or other BTK inhibitor. In an embodiment of the invention, patients pretreated with ibrutinib or other BTK inhibitor have higher fold-expansion as compared with untreated patients.

[0365] In an embodiment of the invention, the method includes a step of adding a BTK inhibitor to the cell culture. In an embodiment, the BTK inhibitor is added on one or more of Day 0, Day 1, Day 2, Day 3, Day 4, Day 5, Day 6, Day 7, Day 8, Day 9, Day 10, Day 11, Day 12, Day 13, or Day 14 of the process. In an embodiment, the BTK inhibitor is added on the days during the method when cell culture medium is exchanged. In an embodiment, the BTK inhibitor is added on Day 0 and when cell culture medium is exchanged. In an embodiment, the BTK inhibitor is added during the method when IL-2 is added. In an embodiment, the BTK inhibitor is added on Day 0, Day 4, Day 7, and optionally Day 11 of the method. In an embodiment of the invention, the BTK inhibitor is added at Day 0 and at Day 7 of the method. In an embodiment of the invention, the BTK inhibitor is one known in the art. In an embodiment of the invention, the BTK inhibitor is one described elsewhere herein.

[0366] In an embodiment of the invention, the BTK inhibitor is used in the method at a concentration of from about 0.1 nM to about 5 uM. In an embodiment, the BTK inhibitor is used in the method at a concentration of about 0.1 nM, 0.5 nM, 1 nM, 5 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, 400 nM, 450 nM, 500 nM, 550 nM, 600 nM, 650 nM, 700 nM, 750 nM, 800 nM, 850 nM, 900 nM, 950 nM, 1 uM, 2 uM, 3 uM, 4 uM, or 5 uM.

[0367] In an embodiment of the invention, the method includes a step of adding a BTK inhibitor when the PBMCs are derived from a patient who has no prior exposure to a BTK inhibitor treatment, such as ibrutinib.

[0368] In some embodiments, the PBMC sample is from a subject or patient who has been optionally pre-treated with a regimen comprising a kinase inhibitor or a BTK inhibitor. In some embodiments, the tumor sample is from a subject or patient who has been pre-treated with a regimen comprising a kinase inhibitor or a BTK inhibitor. In some embodiments, the PBMC sample is from a subject or patient who has been pre-treated with a regimen comprising a kinase inhibitor or a BTK inhibitor, has undergone treatment for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, or 1 year or more. In another embodiment, the PBMCs are derived from a patient who is currently on a BTK inhibitor regimen, such as ibrutinib.

[0369] In some embodiments, the PBMC sample is from a subject or patient who has been pre-treated with a regimen comprising a kinase inhibitor or a BTK inhibitor and is refractory to treatment with a kinase inhibitor or a BTK inhibitor, such as ibrutinib.

[0370] In some embodiments, the PBMC sample is from a subject or patient who has been pre-treated with a regimen comprising a kinase inhibitor or a BTK inhibitor but is no longer undergoing treatment with a kinase inhibitor or a BTK inhibitor. In some embodiments, the PBMC sample is from a subject or patient who has been pre-treated with a regimen comprising a kinase inhibitor or a BTK inhibitor but is no longer undergoing treatment with a kinase inhibitor or a BTK inhibitor and has not undergone treatment for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, or at least 1 year or more. In another embodiment, the PBMCs are derived from a patient who has prior exposure to a BTK inhibitor, but has not been treated in at least 3 months, at least 6 months, at least 9 months, or at least 1 year.

[0371] In an embodiment of the invention, the PBMCs are obtained from a patient who has been treated with a BCL2 inhibitor, such as venetoclax, obatoclax, subatoclax, maritoclax, gossypol, apogossypol, TW-37, UMI-77, BDA-366, navitoclax, and ABT-737. The BCL2 inhibitors are described in further detail by Gupta et al. in “BCL2 Family Inhibitors in the Biology and Treatment of Multiple Myeloma,” Blood and Lymphatic Cancer: Targets and Therapy 2021:11 11-24, which is incorporated herein by reference in its entirety.

[0372] In an embodiment of the invention, the PBMCs are obtained from a patient who has been treated with a BCL2 inhibitor as described elsewhere herein, prior to obtaining a PBMC sample for use with any of the foregoing methods, including PBL Method 1. In an embodiment of the invention, the BCL2 inhibitor treatment has been administered at least 1 time, at least 2, times, or at least 3 times or more.

[0373] In an embodiment of the invention, the method includes a step of adding a BCL2 inhibitor to the cell culture. In an embodiment, the BCL2 inhibitor is added on one or more of Day 0, Day 1, Day 2, Day 3, Day 4, Day 5, Day 6, Day 7, Day 8, Day 9, Day 10, Day 11, Day 12, Day 13, or Day 14 of the process. In an embodiment, the BCL2 inhibitor is added on the days during the method when cell culture medium is exchanged. In an embodiment, the BCL2 inhibitor is added on Day 0 and when cell culture medium is exchanged. In an embodiment, the BCL2 inhibitor is added during the method when IL-2 is added. In an embodiment, the BCL2 inhibitor is added on Day 0, Day 4, Day 7, and optionally Day 11 of the method. In an embodiment of the invention, the BCL2 inhibitor is added at Day 0 and at Day 7 of the method. In an embodiment of the invention, the BCL2 inhibitor is one known in the art. In an embodiment of the invention, the BCL2 inhibitor is one described elsewhere herein.

[0374] In an embodiment of the invention, the BCL2 inhibitor is used in the method at a concentration of from about 0.1 nM to about 5 uM. In an embodiment, the BCL2 inhibitor is used in the method at a concentration of about 0.1 nM, 0.5 nM, 1 nM, 5 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, 400 nM, 450 nM, 500 nM, 550 nM, 600 nM, 650 nM, 700 nM, 750 nM, 800 nM, 850 nM, 900 nM, 950 nM, 1 uM, 2 uM, 3 uM, 4 uM, or 5 uM.

[0375] In an embodiment of the invention, the method includes a step of adding a BCL2 inhibitor when the PBMCs are derived from a patient who has no prior exposure to a BCL2 inhibitor treatment, such as venetoclax.

[0376] In some embodiments, the PBMC sample is from a subject or patient who has been optionally pre-treated with a regimen comprising a BCL2 inhibitor. In some embodiments, the tumor sample is from a subject or patient who has been pre-treated with a regimen comprising a BCL2 inhibitor. In some embodiments, the PBMC sample is from a subject or patient who has been pre-treated with a regimen comprising a BCL2 inhibitor, has undergone treatment for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, or 1 year or more. In another embodiment, the PBMCs are derived from a patient who is currently on a BCL2 inhibitor, such as venetoclax.

[0377] In some embodiments, the PBMC sample is from a subject or patient who has been pre-treated with a regimen comprising a kinase inhibitor or a BCL2 inhibitor and is refractory to treatment with a kinase inhibitor or a BCL2 inhibitor, such as venetoclax.

[0378] In some embodiments, the PBMC sample is from a subject or patient who has been pre-treated with a regimen comprising a BCL2 inhibitor but is no longer undergoing treatment with a BCL2 inhibitor. In some embodiments, the PBMC sample is from a subject or patient who has been pre-treated with a regimen comprising a BCL2 inhibitor but is no longer undergoing treatment with a BCL2 inhibitor and has not undergone treatment for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, or at least 1 year or more. In another embodiment, the PBMCs are derived from a patient who has prior exposure to a BCL2 inhibitor, but has not been treated in at least 3 months, at least 6 months, at least 9 months, or at least 1 year.

[0379] In some embodiments, the PBMCs are obtained from a patient who has been treated with a combination of an ITK inhibitor, a BKT inhibitor and / or a BCL2 inhibitor as described elsewhere herein, prior to obtaining a PBMC sample for use with any of the foregoing methods, including PBL Method 1. In an embodiment of the invention, the combination treatment has been administered at least 1 time, at least 2, times, or at least 3 times or more.

[0380] In an embodiment of the invention, the method includes a step of adding a combination of an ITK inhibitor, a BKT inhibitor and / or a BCL2 inhibitor to the cell culture. In an embodiment, the combination of an ITK inhibitor, a BKT inhibitor and / or a BCL2 inhibitor is added on one or more of Day 0, Day 1, Day 2, Day 3, Day 4, Day 5, Day 6, Day 7, Day 8, Day 9, Day 10, Day 11, Day 12, Day 13, or Day 14 of the process. In an embodiment, the combination of an ITK inhibitor, a BKT inhibitor and / or a BCL2 inhibitor is added on the days during the method when cell culture medium is exchanged. In an embodiment, the combination of an ITK inhibitor, a BKT inhibitor and / or a BCL2 inhibitor is added on Day 0 and when cell culture medium is exchanged. In an embodiment, the combination of an ITK inhibitor, a BKT inhibitor and / or a BCL2 inhibitor is added during the method when IL-2 is added. In an embodiment, the combination of an ITK inhibitor, a BKT inhibitor and / or a BCL2 inhibitor is added on Day 0, Day 4, Day 7, and optionally Day 11 of the method. In an embodiment of the invention, the combination of an ITK inhibitor, a BKT inhibitor and / or a BCL2 inhibitor is added at Day 0 and at Day 7 of the method. In an embodiment of the invention, the ITK inhibitor, the BKT inhibitor and / or the BCL2 inhibitor is one known in the art.

[0381] In an embodiment of the invention, the combination of an ITK inhibitor, a BKT inhibitor and / or a BCL2 inhibitor is used in the method at a concentration of from about 0.1 nM to about 5 uM. In an embodiment, the combination of an ITK inhibitor, a BKT inhibitor and / or a BCL2 inhibitor is used in the method at a concentration of about 0.1 nM, 0.5 nM, 1 nM, 5 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, 400 nM, 450 nM, 500 nM, 550 nM, 600 nM, 650 nM, 700 nM, 750 nM, 800 nM, 850 nM, 900 nM, 950 nM, 1 uM, 2 uM, 3 uM, 4 uM, or 5 uM.

[0382] In an embodiment of the invention, the method includes a step of adding a combination of an ITK inhibitor, a BKT inhibitor and / or a BCL2 inhibitor when the PBMCs are derived from a patient who has no prior exposure to an ITK inhibitor, a BKT inhibitor and / or a BCL2 inhibitor treatment.

[0383] In some embodiments, the PBMC sample is from a subject or patient who has been optionally pre-treated with a regimen comprising a combination of an ITK inhibitor, a BKT inhibitor and / or a BCL2 inhibitor. In some embodiments, the tumor sample is from a subject or patient who has been pre-treated with a regimen comprising a combination of an ITK inhibitor, a BKT inhibitor and / or a BCL2 inhibitor. In some embodiments, the PBMC sample is from a subject or patient who has been pre-treated with a regimen comprising a combination of an ITK inhibitor, a BKT inhibitor and / or a BCL2 inhibitor, has undergone treatment for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, or 1 year or more. In another embodiment, the PBMCs are derived from a patient who is currently on a combination of an ITK inhibitor, a BKT inhibitor and / or a BCL2 inhibitor.

[0384] In some embodiments, the PBMC sample is from a subject or patient who has been pre-treated with a regimen comprising a combination of an ITK inhibitor, a BKT inhibitor and / or a BCL2 inhibitor and is refractory to treatment with the combination of an ITK inhibitor, a BKT inhibitor and / or a BCL2 inhibitor.

[0385] In some embodiments, the PBMC sample is from a subject or patient who has been pre-treated with a regimen comprising a combination of an ITK inhibitor, a BKT inhibitor and / or a BCL2 inhibitor but is no longer undergoing treatment with a combination of an ITK inhibitor, a BKT inhibitor and / or a BCL2 inhibitor. In some embodiments, the PBMC sample is from a subject or patient who has been pre-treated with a regimen comprising a combination of an ITK inhibitor, a BKT inhibitor and / or a BCL2 inhibitor but is no longer undergoing treatment with the combination of an ITK inhibitor, a BKT inhibitor and / or a BCL2 inhibitor and has not undergone treatment for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, or at least 1 year or more. In another embodiment, the PBMCs are derived from a patient who has prior exposure to the combination of an ITK inhibitor, a BKT inhibitor and / or a BCL2 inhibitor, but has not been treated in at least 3 months, at least 6 months, at least 9 months, or at least 1 year.

[0386] In an embodiment of the invention, at Day 0, cells are selected for CD19+ and sorted accordingly. In an embodiment of the invention, the selection is made using antibody binding beads. In an embodiment of the invention, pure T-cells are isolated on Day 0 from the PBMCs. In an embodiment of the invention, at Day 0, the CD19+ B-cells and pure T-cells are co-cultured with antiCD3 / antiCD28 antibodies for a minimum of 4 days. In an embodiment of the invention, on Day 4, IL-2 is added to the culture. In an embodiment of the invention, on Day 7, the culture is restimulated with antiCD3 / antiCD28 antibodies and additional IL-2. In an embodiment of the invention, on Day 14, the PBLs are harvested.

[0387] In an embodiment of the invention, for patients that are not pre-treated with ibrutinib or other ITK inhibitor, 10-15 ml of Buffy Coat will yield about 5×109 PBMC, which, in turn, will yield about 5.5×107 starting cell material, and about 11×109 PBLs at the end of the expansion process. In an embodiment of the invention, about 54×106 PBMCs will yield about 6×105 starting material, and about 1.2×108 MIL (about a 205-fold expansion).

[0388] In an embodiment of the invention, for patients that are pre-treated with ibrutinib or other ITK inhibitor, the expansion process will yield about 20×109 PBLs. In an embodiment of the invention, 40.3×106 PBMCs will yield about 4.7×105 starting cell material, and about 1.6×108 PBLs (about a 338-fold expansion).

[0389] In an embodiment of the invention, the clinical dose of PBLs useful in the present invention for patients with chronic lymphocytic leukemia (CLL) is from about 0.1×109 to about 15×109 PBLs, from about 0.1×109 to about 15×109 PBLs, from about 0.12×109 to about 12×109 PBLs, from about 0.15×109 to about 11×109 PBLs, from about 0.2×109 to about 10×109 PBLs, from about 0.3×109 to about 9×109 PBLs, from about 0.4×109 to about 8×109 PBLs, from about 0.5×109 to about 7×109 PBLs, from about 0.6×109 to about 6×109 PBLs, from about 0.7×109 to about 5×109 PBLs, from about 0.8×109 to about 4×109 PBLs, from about 0.9×109 to about 3×109 PBLs, or from about 1×109 to about 2×109 PBLs.

[0390] In any of the foregoing embodiments, PBMCs may be derived from a whole blood sample, by apheresis, from the buffy coat, or from any other method known in the art for obtaining PBMCs.

[0391] In an embodiment, the invention provides a method for the preparation of peripheral blood lymphocytes (PBLs) comprising the steps of:

[0392] a. Obtaining a sample of peripheral blood mononuclear cells (PBMCs) from the peripheral blood of a patient, wherein said sample is optionally cryopreserved and the patient is optionally pretreated with an ITK inhibitor;

[0393] b. Optionally washing the PBMCs by centrifugation;

[0394] c. Admixing magnetic beads selective for CD3 and CD28 to the PBMCs to form an admixture of the beads and the PBMCs;

[0395] d. Seeding the admixture of the beads and the PBMCs into a gas-permeable container and co-culturing said PBMCs in media comprising about 3000 IU / mL of IL-2 in for about 4 to about 6 days;

[0396] e. Feeding said PBMCs using media comprising about 3000 IU / mL of IL-2, and co-culturing said PBMCs for about 5 days, such that the total co-culture period of steps d and e is about 9 to about 11 days;

[0397] f. Harvesting PBMCs from media;

[0398] g. Removing the magnetic beads selective for CD3 and CD28 from the harvested PBMCs using a magnet;

[0399] h. Removing residual B-cells from the harvested PBMCs using magnetic-activated cell sorting and magnetic beads selective for CD19 to provide a PBL product;

[0400] i. Washing and concentrating the PBL product using a cell harvester; and

[0401] j. Formulating and optionally cryopreserving the PBL product,wherein the ITK inhibitor is optionally an ITK inhibitor that covalently binds to ITK.

[0402] In an embodiment, the invention provides a method for the preparation of peripheral blood lymphocytes (PBLs) from a whole blood sample, the method comprising the steps of:

[0403] (a) obtaining peripheral blood mononuclear cells (PBMCs) from less than or equal to about 50 mL of whole blood from a patient having a liquid tumor, wherein the patient is optionally pretreated with an ITK inhibitor;

[0404] (b) admixing beads selective for CD3 and CD28 with the PBMCs, wherein the beads are added at a ratio of 3 beads:1 cell, to form an admixture of the PBMCs and the beads;

[0405] (c) culturing the admixture of the PBMCs and the beads at a density of about 25,000 cells per cm2 to about 50,000 cells per cm2 on a gas-permeable surface of one or more containers containing a first cell culture medium and IL-2 for a period of about 4 days;

[0406] (d) adding to each container of step (c) IL-2 and a second cell culture medium that is the same as or different from the first cell culture medium and culturing for a period of about 5 days to about 7 days to form an expanded population of PBLs; and

[0407] (e) harvesting from each container the expanded population of PBLs.

[0408] In an embodiment, the invention provides a method for the preparation of peripheral blood lymphocytes (PBLs) from a whole blood sample, the method comprising the steps of:

[0409] (a) obtaining peripheral blood mononuclear cells (PBMCs) from less than or equal to about 50 mL of whole blood from a patient having a liquid tumor, wherein the patient is optionally pretreated with an ITK inhibitor;

[0410] (b) removing B-cells from the PBMCs by selecting against CD19 to provide PBMCs depleted of B-cells;

[0411] (c) admixing beads selective for CD3 and CD28 with the PBMCs, wherein the beads are added at a ratio of 3 beads:1 cell, to form an admixture of the PBMCs and the beads;

[0412] (d) culturing the admixture of the PBMCs and the beads at a density of about 25,000 cells per cm2 to about 50,000 cells per cm2 on a gas-permeable surface of one or more containers containing a first cell culture medium and IL-2 for a period of about 4 days;

[0413] (e) adding to each container of step (d) IL-2 and a second cell culture medium that is the same as or different from the first cell culture medium and culturing for a period of about 5 days to about 7 days to form an expanded population of PBLs; and

[0414] (f) harvesting from each container the expanded population of PBLs.

[0415] In an embodiment, the invention provides a method for the preparation of peripheral blood lymphocytes (PBLs) from a whole blood sample, the method comprising the steps of:

[0416] (a) obtaining peripheral blood mononuclear cells (PBMCs) from less than or equal to about 50 mL of whole blood from a patient having a liquid tumor, wherein the patient is optionally pretreated with an ITK inhibitor;

[0417] (b) determining the proportion of the PMBCs constituted by B-cells as a B-cell percentage;

[0418] (c) if the B-cell percentage determined in step (b) is at least about seventy percent (70%), removing B-cells from the PBMCs by selecting against CD19 to provide PBMCs depleted of B-cells;

[0419] (d) admixing beads selective for CD3 and CD28 with the PBMCs, wherein the beads are added at a ratio of 3 beads:1 cell, to form an admixture of the PBMCs and the beads;

[0420] (e) culturing the admixture of the PBMCs and the beads at a density of about 25,000 cells per cm2 to about 50,000 cells per cm2 on a gas-permeable surface of one or more containers containing a first cell culture medium and IL-2 for a period of about 4 days;

[0421] (f) adding to each container of step (d) IL-2 and a second cell culture medium that is the same as or different from the first cell culture medium and culturing for a period of about 5 days to about 7 days to form an expanded population of PBLs; and

[0422] (g) harvesting from each container the expanded population of PBLs.

[0423] In an embodiment of the invention, removal of B-cells, or B-cell depletion (BCD), occurs on Day 0 or on Day 9 of a 9-day expansion process. In another embodiment, the BCD occurs on both Day 0 and Day 9 of a 9-day expansion process. In an embodiment of the invention, BCD occurs on Day 0 or Day 11 of an 11-day expansion process. In another embodiment, the BCD occurs on both Day 0 and Day 11 of an 11-day expansion process.

[0424] In an embodiment of the invention, the BCD step is performed on a PBMC sample from a patient having a high initial B-cell count. In one embodiment, a high initial B-cell count is about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more B-cells in the initial PBMC sample.

[0425] In an embodiment, the invention provides any of the methods described above modified as applicable such that if the B-cell percentage is at least about 70% the B-cell removal step, or BCD step, is performed.

[0426] In an embodiment, the invention provides any of the methods described above modified as applicable such that if the B-cell percentage is at least about 75% the B-cell removal step is performed.

[0427] In an embodiment, the invention provides any of the methods described above modified as applicable such that if the B-cell percentage is at least about 80% the B-cell removal step is performed.

[0428] In an embodiment, the invention provides any of the methods described above modified as applicable such that if the B-cell percentage is at least about 85% the B-cell removal step is performed.

[0429] In an embodiment, the invention provides any of the methods described above modified as applicable such that if the B-cell percentage is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more the B-cell removal step is performed.

[0430] In an embodiment, the invention provides any of the methods described above modified as applicable such that the B-cell percentage is determined by comparison of the CD19+ cells to the CD45+ cells in the PBMCs.

[0431] In an embodiment, the invention provides any of the methods described above modified as applicable such that the B-cell percentage is determined by comparison of the fraction of CD19+ / CD45+ cells to the fraction of CD45+ cells in the PBMCs.

[0432] In an embodiment, the invention provides any of the methods described above modified as applicable such that the comparison of the fraction of CD19+ cells to the fraction of CD45+ cells in the PBMCs is performed by contacting the PBMCs with a CD19 stain and a CD45 stain, and then comparing the subpopulation of PBMCs positive for the both CD19 stain and the CD45 stain with the subpopulation of PBMCs positive for only the CD19 stain.

[0433] In an embodiment, the invention provides any of the methods described above modified as applicable such that the CD19 stain is an anti-CD19 antibody conjugated to a first label and the CD45 stain is an anti-CD45 antibody conjugated to a second label.

[0434] In an embodiment, the invention provides any of the methods described above modified as applicable such that the first label is a first fluorochrome and the second label is a second fluorochrome that is different from the first fluorochrome.

[0435] In an embodiment, the invention provides any of the methods described above modified as applicable such that the total culturing period is from at or about 9 days to at or about 11 days.

[0436] In an embodiment, the invention provides any of the methods described above modified as applicable such that the total culturing period is at or about 9 days, at or about 10 days or at or about 11 days.

[0437] In an embodiment, the invention provides any of the methods described above modified as applicable such that the total culturing period is from at or about 9 days to at or about 14 days.

[0438] In an embodiment, the invention provides any of the methods described above modified as applicable such that the total culturing period is at or about 9 days, at or about 10 days, at or about 11 days, at or about 12 days, at or about 13 days, or at or at or about 14 days.

[0439] In an embodiment, the invention provides any of the methods described above modified as applicable such that the PBMCs are obtained from at or about 50 mL of peripheral blood of the patient.

[0440] In an embodiment, the invention provides any of the methods described above modified as applicable such that the PBMCs are obtained from at or about 10 mL to at or about 50 mL of peripheral blood of the patient.

[0441] In an embodiment, the invention provides any of the methods described above modified as applicable such that the PBMCs are obtained from at or about 10 mL, at or about 20 mL, at or about 30 mL, at or about 40 mL, or at or about 50 mL of peripheral blood of the patient.

[0442] In an embodiment, the invention provides any of the methods described above modified as applicable such that the PBMCs are obtained from at or about 10 mL to at or about 100 mL of peripheral blood of the patient

[0443] In an embodiment, the invention provides any of the methods described above modified as applicable such that the PBMCs are obtained from at or about 10 mL, at or about 20 mL, at or about 30 mL, at or about 40 mL, at or about 50 mL, at or about 60 mL, at or about 70 mL, at or about 80 mL, at or about 90 mL, or at or about 100 mL of peripheral blood of the patient.

[0444] In an embodiment, the invention provides any of the methods described above modified as applicable such that the total number of cells harvested is from at or about 1 billion to at or about 8 billion.

[0445] In an embodiment, the invention provides any of the methods described above modified as applicable such that the total number of cells harvested is from at or about 1 billion, about 2 billion, about 3 billion, about 4 billion, about 5 billion, and 6 billion, about 7 billion, about 8 billion, about 9 billion, or about 10 billion.

[0446] In an embodiment, the invention provides any of the methods described above modified as applicable such that the total number of cells harvested is from at or about 8 billion to at or about 22 billion.

[0447] In an embodiment, the invention provides any of the methods described above modified as applicable such that the total number of cells harvested is from at or about 2 billion to at or about 50 billion.

[0448] In an embodiment, the invention provides any of the methods described above modified as applicable such that the total number of cells harvested is from at or about 8 billion, at or about 9 billion, at or about 10 billion, at or about 11 billion, at or about 12 billion, at or about 13 billion, at or about 14 billion, at or about 15 billion, at or about 16 billion, at or about 17 billion, at or about 18 billion, at or about 19 billion, at or about 20 billion, at or about 21 billion, or at or about 22 billion.

[0449] In an embodiment, the invention provides any of the methods described above modified as applicable such that the PBMCs are cultured in a plurality of gas-permeable containers.

[0450] In an embodiment, the invention provides any of the methods described above modified as applicable such that the PBMCs are cultured in at least two gas-permeable containers.

[0451] In an embodiment, the invention provides any of the methods described above modified as applicable such that the PBMCs are cultured in at least five gas-permeable containers.

[0452] In an embodiment, the invention provides any of the methods described above modified as applicable such that the PBMCs are cultured in 2 to 20 gas-permeable containers.

[0453] In an embodiment, the invention provides any of the methods described above modified as applicable such that the PBMCs are cultured in up to 5 gas-permeable containers.

[0454] In an embodiment, the invention provides any of the methods described above modified as applicable such that the PBMCs are cultured in 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 gas-permeable containers.

[0455] In an embodiment, the invention provides any of the methods described above modified as applicable such that the PBMCs are seeded at a density of at or about 12,500 cells per cm2 to at or about 50,000 cells per cm2 in each gas-permeable container.

[0456] In an embodiment, the invention provides any of the methods described above modified as applicable such that the PBMCs are seeded at a density of at or about 6,250 cells per cm2 to at or about 25,000 cells per cm2 in each gas-permeable container.

[0457] In an embodiment, the invention provides any of the methods described above modified as applicable such that the PBMCs are seeded at a density of at or about 6,250 cells per cm2 to at or about 50,000 cells per cm2 in each gas-permeable container.

[0458] In an embodiment, the invention provides any of the methods described above modified as applicable such that the PBMCs are seeded at a density of at or about 25,000 cells per cm2 to at or about 50,000 cells per cm2 in each gas-permeable container.

[0459] In an embodiment, the invention provides any of the methods described above modified as applicable such that the PBMCs are seeded at a density of at or about 6,250 cells per cm2, at or about 9,375 cells per cm2, at or about 12,500 cells per cm2, at or about 15,625 cells per cm2, at or about 18,750 cells per cm2, at or about 21,875 cells per cm2, at or about 25,000 cells per cm2, at or about 28,125 cells per cm2, at or about 31,250 cells per cm2, at or about 34,375 cells per cm2, at or about 37,500 cells per cm2, at or about 40,625 cells per cm2, at or about 43,750 cells per cm2, at or about 47,875 cells per cm2, or at or about at or about 50,000 cells per cm2 in each gas-permeable container.

[0460] In an embodiment, the invention provides any of the methods described above modified as applicable such that the step of admixing the beads selective for CD3 and CD28 with the PBMCs to form an admixture of the beads and the PBMCs is replaced with the step of admixing the beads selective for CD3 and CD28 with the PBMCs to form complexes of the beads and the PBMCs in an admixture of the beads and the PBMCs, and wherein the step of culturing the admixture is replaced with the step of separating the complexes of the beads and the PBMCs from the admixture and culturing the complexes of PBMCs and the beads at a density of about 25,000 cells per cm2 to about 50,000 cells per cm2 on a gas-permeable surface in one or more containers containing a first cell culture medium and IL-2 for a period of about 4 days. In another embodiment, the beads selective for CD3 and CD28 are magnetic beads, and the step of separating the complexes of the beads and the PBMCs from the admixture is performed by using a magnet to remove the complexes from the admixture.

[0461] In an embodiment, the invention provides any of the methods described above modified as applicable such that the beads selective for CD3 and CD28 are beads conjugated to anti-CD3 antibodies and anti-CD28 antibodies.

[0462] In an embodiment, the invention provides any of the methods described above modified as applicable such that the removal of B-cells from the PBMCs is performed by contacting PBMCs with beads selective for CD19 to form bead-CD19+ cell complexes and removing the complexes to provide PBMCs depleted of B-cells. In another embodiment, the beads selective for CD19 are magnetic beads and a magnet is used to remove magnetic bead-CD19+ cell complexes from the PBMCs. In another embodiment, the beads selective for CD19 are beads conjugated to anti-CD19 antibodies. In another embodiment, the beads conjugated to anti-CD19 antibodies are CliniMACS™ anti-CD19 beads (Miltenyi).

[0463] In an embodiment, the invention provides any of the methods described above modified as applicable such that after the step of harvesting the expanded population of PBLs the method comprises the step of performing a selection to remove any remnant B-cells from the expanded population of PBLs.

[0464] In an embodiment, the invention provides any of the methods described above modified as applicable such that the selection to remove any remnant B-cells from the expanded population of PBLs is performed by admixing beads selective for CD19 with the expanded population of PBLs to form complexes of beads and any remnant B-cells and removing the complexes from the expanded population of PBLs.

[0465] In an embodiment, the invention provides any of the methods described above modified as applicable such that the selection to remove any remnant B-cells from the expanded population of PBLs is performed by admixing magnetic beads selective for CD19 with the expanded population of PBLs to form complexes of magnetic beads and any remnant B-cells and using a magnet to remove the complexes from the expanded population of PBLs.

[0466] In an embodiment, the invention provides any of the methods described above modified as applicable such that the beads selective for CD19 are beads conjugated to anti-CD19 antibody.

[0467] In an embodiment, the invention provides any of the methods described above modified as applicable such that the first cell culture medium contains about 3000 IU / mL of IL-2.

[0468] In an embodiment, the invention provides any of the methods described above modified as applicable such that the second cell culture medium contains about 3000 IU / mL of IL-2.

[0469] In an embodiment, the invention provides any of the methods described above modified as applicable such that the cultures in the culturing steps are incubated at 37° C. and under an atmosphere containing 5% CO2.

[0470] In an embodiment, the invention provides any of the methods described above modified as applicable such that the patient is pretreated with an ITK inhibitor.

[0471] In an embodiment, the invention provides any of the methods described above modified as applicable such that the patient is pretreated with an ITK inhibitor and is refractory to treatment with the ITK inhibitor.

[0472] In an embodiment, the invention provides any of the methods described above modified as applicable such that the patient is pretreated with ibrutinib.

[0473] In an embodiment, the invention provides any of the methods described above modified as applicable such that the patient is suffering from a leukemia.

[0474] In an embodiment, the invention provides any of the methods described above modified as applicable such that the patient is suffering from a chronic lymphocytic leukemia.Pharmaceutical Compositions, Dosages, and Dosing Regimens for PBLs

[0475] In another embodiment, the invention provides a therapeutic population of PBLs prepared by any method of expanding PBLs described herein, optionally modified to express a chimeric antigen receptor (CAR) and / or express a modified T-cell receptor and / or suppress or reduce expression of one or more immune checkpoint genes as described herein.

[0476] In another embodiment, the invention provides a pharmaceutical composition comprising a therapeutic population of PBLs prepared by any method of expanding PBLs described herein, optionally modified to express a chimeric antigen receptor (CAR) and / or express a modified T-cell receptor and / or suppress or reduce expression of one or more immune checkpoint genes as described herein, and a pharmaceutically acceptable carrier.

[0477] In an embodiment, PBLs expanded using methods of the present disclosure are administered to a patient as a pharmaceutical composition. In an embodiment, the pharmaceutical composition is a suspension of PBLs in a sterile buffer. PBLs expanded using methods of the present disclosure may be administered by any suitable route as known in the art. Preferably, the PBLs are administered as a single intra-arterial or intravenous infusion, which preferably lasts approximately 30 to 60 minutes. Other suitable routes of administration include intraperitoneal, intrathecal, and intralymphatic administration.

[0478] Any suitable dose of PBLs can be administered. Preferably, from about 2.3×1010 to about 13.7×1010 PBLs are administered, with an average of around 7.8×1010 PBLs, particularly if the cancer is a hematological malignancy. In an embodiment, about 1.2×1010 to about 4.3×1010 of PBLs are administered. In an embodiment, about 8 billion to about 22 billion PBLs are administered.

[0479] In some embodiments, the number of the PBLs provided in the pharmaceutical compositions of the invention is about 1×106, 2×106, 3×106, 4×106, 5×106, 6×106, 7×106, 8×106, 9×106, 1×107, 2×107, 3×107, 4×107, 5×107, 6×107, 7×107, 8×107, 9×107, 1×108, 2×108, 3×108, 4×108, 5×108, 6×108, 7×108, 8×108, 9×108, 1×109, 2×109, 3×109, 4×109, 5×109, 6×109, 7×109, 8×109, 9×109, 1×1010, 2×1010, 3×1010, 4×1010, 5×1010, 6×1010, 7×1010, 8×1010, 9×1010, 1×1011, 2×1011, 3×1011, 4×1011, 5×1011, 6×1011, 7×1011, 8×1011, 9×1011, 1×1012, 2×1012, 3×1012, 4×1012, 5×1012, 6×1012, 7×1012 8×1012, 9×1012, 1×1013, 2×1013, 3×1013, 4×1013, 5×1013, 6×1013, 7×1013, 8×103, and 9×1013. In an embodiment, the number of the PBLs provided in the pharmaceutical compositions of the invention is in the range of 1×106 to 5×106, 5×106 to 1×107, 1×107 to 5×107, 5×107 to 1×108, 1×108 to 5×108, 5×108 to 1×109, 1×109 to 5×109, 5×109 to 1×1010, 1×1010 to 5×1010, 5×1010 to 1×1011, 5×1011 to 1×1011, 1×1012 to 5×1012, and 5×1012 to 1×1013. In an embodiment of the invention, the number of PBLs provided in the pharmaceutical compositions of the invention is in the range of from about 4×108 to about 2.5×109. In another embodiment, the number of PBLs provided in the pharmaceutical compositions of the invention is 9.5×108. In another embodiment, the number of PBLs provided in the pharmaceutical compositions of the invention is 4.1×108. In another embodiment, the number of PBLs provided in the pharmaceutical compositions of the invention is 2.2×109.

[0480] In an embodiment of the invention, the number of PBLs provided in the pharmaceutical compositions of the invention is in the range of from about 0.1×109 to about 15×109 PBLs, from about 0.1×109 to about 15×109 PBLs, from about 0.12×109 to about 12×109 PBLs, from about 0.15×109 to about 11×109 PBLs, from about 0.2×109 to about 10×109 PBLs, from about 0.3×109 to about 9×109 PBLs, from about 0.4×109 to about 8×109 PBLs, from about 0.5×109 to about 7×109 PBLs, from about 0.6×109 to about 6×109 PBLs, from about 0.7×109 to about 5×109 PBLs, from about 0.8×109 to about 4×109 PBLs, from about 0.9×109 to about 3×109 PBLs, or from about 1×109 to about 2×109 PBLs.

[0481] In some embodiments, the concentration of the PBLs provided in the pharmaceutical compositions of the invention is less than, for example, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17% 16%, 15%, 14%, 13%, 12% 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001% w / w, w / v or v / v of the pharmaceutical composition.

[0482] In some embodiments, the concentration of the PBLs provided in the pharmaceutical compositions of the invention is greater than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19.75%, 19.50%, 19.25% 19%, 18.75%, 18.50%, 18.25% 18%, 17.75%, 17.50%, 17.25% 17%, 16.75%, 16.50%, 16.25% 16%, 15.75%, 15.50%, 15.25% 15%, 14.75%, 14.50%, 14.25% 14%, 13.75%, 13.50%, 13.25% 13%, 12.75%, 12.50%, 12.25% 12%, 11.75%, 11.50%, 11.25% 11%, 10.75%, 10.50%, 10.25% 10%, 9.75%, 9.50%, 9.25% 9%, 8.75%, 8.50%, 8.25% 8%, 7.75%, 7.50%, 7.25% 7%, 6.75%, 6.50%, 6.25% 6%, 5.75%, 5.50%, 5.25% 5%, 4.75% 4.50%, 4.25% 4%, 3.75%, 3.50%, 3.25%, 3%, 2.75%, 2.50%, 2.25%, 2%, 1.75%, 1.50%, 125%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001% w / w, w / v, or v / v of the pharmaceutical composition.

[0483] In some embodiments, the concentration of the PBLs provided in the pharmaceutical compositions of the invention is in the range from about 0.0001% to about 50%, about 0.001% to about 40%, about 0.01% to about 30%, about 0.02% to about 29%, about 0.03% to about 28%, about 0.04% to about 27%, about 0.05% to about 26%, about 0.06% to about 25%, about 0.07% to about 24%, about 0.08% to about 23%, about 0.09% to about 22%, about 0.1% to about 21%, about 0.2% to about 20%, about 0.3% to about 19%, about 0.4% to about 18%, about 0.5% to about 17%, about 0.6% to about 16%, about 0.7% to about 15%, about 0.8% to about 14%, about 0.9% to about 12% or about 1% to about 10% w / w, w / v or v / v of the pharmaceutical composition.

[0484] In some embodiments, the concentration of the PBLs provided in the pharmaceutical compositions of the invention is in the range from about 0.001% to about 10%, about 0.01% to about 5%, about 0.02% to about 4.5%, about 0.03% to about 4%, about 0.04% to about 3.5%, about 0.05% to about 3%, about 0.06% to about 2.5%, about 0.07% to about 2%, about 0.08% to about 1.5%, about 0.09% to about 1%, about 0.1% to about 0.9% w / w, w / v or v / v of the pharmaceutical composition.

[0485] In some embodiments, the amount of the PBLs provided in the pharmaceutical compositions of the invention is equal to or less than 10 g, 9.5 g, 9.0 g, 8.5 g, 8.0 g, 7.5 g, 7.0 g, 6.5 g, 6.0 g, 5.5 g, 5.0 g, 4.5 g, 4.0 g, 3.5 g, 3.0 g, 2.5 g, 2.0 g, 1.5 g, 1.0 g, 0.95 g, 0.9 g, 0.85 g, 0.8 g, 0.75 g, 0.7 g, 0.65 g, 0.6 g, 0.55 g, 0.5 g, 0.45 g, 0.4 g, 0.35 g, 0.3 g, 0.25 g, 0.2 g, 0.15 g, 0.1 g, 0.09 g, 0.08 g, 0.07 g, 0.06 g, 0.05 g, 0.04 g, 0.03 g, 0.02 g, 0.01 g, 0.009 g, 0.008 g, 0.007 g, 0.006 g, 0.005 g, 0.004 g, 0.003 g, 0.002 g, 0.001 g, 0.0009 g, 0.0008 g, 0.0007 g, 0.0006 g, 0.0005 g, 0.0004 g, 0.0003 g, 0.0002 g, or 0.0001 g.

[0486] In some embodiments, the amount of the PBLs provided in the pharmaceutical compositions of the invention is more than 0.0001 g, 0.0002 g, 0.0003 g, 0.0004 g, 0.0005 g, 0.0006 g, 0.0007 g, 0.0008 g, 0.0009 g, 0.001 g, 0.0015 g, 0.002 g, 0.0025 g, 0.003 g, 0.0035 g, 0.004 g, 0.0045 g, 0.005 g, 0.0055 g, 0.006 g, 0.0065 g, 0.007 g, 0.0075 g, 0.008 g, 0.0085 g, 0.009 g, 0.0095 g, 0.01 g, 0.015 g, 0.02 g, 0.025 g, 0.03 g, 0.035 g, 0.04 g, 0.045 g, 0.05 g, 0.055 g, 0.06 g, 0.065 g, 0.07 g, 0.075 g, 0.08 g, 0.085 g, 0.09 g, 0.095 g, 0.1 g, 0.15 g, 0.2 g, 0.25 g, 0.3 g, 0.35 g, 0.4 g, 0.45 g, 0.5 g, 0.55 g, 0.6 g, 0.65 g, 0.7 g, 0.75 g, 0.8 g, 0.85 g, 0.9 g, 0.95 g, 1 g, 1.5 g, 2 g, 2.5, 3 g, 3.5, 4 g, 4.5 g, 5 g, 5.5 g, 6 g, 6.5 g, 7 g, 7.5 g, 8 g, 8.5 g, 9 g, 9.5 g, or 10 g.

[0487] The PBLs provided in the pharmaceutical compositions of the invention are effective over a wide dosage range. The exact dosage will depend upon the route of administration, the form in which the compound is administered, the gender and age of the subject to be treated, the body weight of the subject to be treated, and the preference and experience of the attending physician. The clinically-established dosages of the PBLs may also be used if appropriate. The amounts of the pharmaceutical compositions administered using the methods herein, such as the dosages of PBLs, will be dependent on the human or mammal being treated, the severity of the disorder or condition, the rate of administration, the disposition of the active pharmaceutical ingredients and the discretion of the prescribing physician.

[0488] In some embodiments, PBLs may be administered in a single dose. Such administration may be by injection, e.g., intravenous injection. In some embodiments, PBLs may be administered in multiple doses. Dosing may be once, twice, three times, four times, five times, six times, or more than six times per year. Dosing may be once a month, once every two weeks, once a week, or once every other day. Administration of PBLs may continue as long as necessary.

[0489] In some embodiments, an effective dosage of PBLs is about 1×106, 2×106, 3×106, 4×106, 5×106, 6×106, 7×106 8×106, 9×106, 1×107, 2×107, 3×107, 4×107, 5×107, 6×107, 7×107, 8×107, 9×107, 1×108, 2×108, 3×108, 4×108, 5×108, 6×108, 7×108, 8×108, 9×108, 1×109, 2×109, 3×109, 4×109, 5×109, 6×109, 7×109, 8×109, 9×109, 1×1010, 2×1010, 3×1010, 4×1010, 5×1010, 6×1010, 7×1010, 8×1010, 9×1010, 1×1011, 2×1011, 3×1011 4×1011, 5×1011, 6×1011, 7×1011 8×1011, 9×1011, 1×1012, 2×1012, 3×1012, 4×1012, 5×1012, 6×1012, 7×1012, 8×1012, 9×1012, 1×1013, 2×1013, 3×1013, 4×1013, 5×1013 6×1013, 7×1013, 8×1013, and 9×1013. In some embodiments, an effective dosage of PBLs is in the range of 1×106 to 5×106, 5×106 to 1×107, 1×107 to 5×107, 5×107 to 1×108, 1×108 to 5×108, 5×108 to 1×109, 1×109 to 5×109, 5×109 to 1×1010, 1×1010 to 5×1010, 5×1010 to 1×1011, 5×1011 to 1×1012, 1×1012 to 5×1012, and 5×1012 to 1×103.

[0490] In some embodiments, an effective dosage of PBLs is in the range of about 0.01 mg / kg to about 4.3 mg / kg, about 0.15 mg / kg to about 3.6 mg / kg, about 0.3 mg / kg to about 3.2 mg / kg, about 0.35 mg / kg to about 2.85 mg / kg, about 0.15 mg / kg to about 2.85 mg / kg, about 0.3 mg to about 2.15 mg / kg, about 0.45 mg / kg to about 1.7 mg / kg, about 0.15 mg / kg to about 1.3 mg / kg, about 0.3 mg / kg to about 1.15 mg / kg, about 0.45 mg / kg to about 1 mg / kg, about 0.55 mg / kg to about 0.85 mg / kg, about 0.65 mg / kg to about 0.8 mg / kg, about 0.7 mg / kg to about 0.75 mg / kg, about 0.7 mg / kg to about 2.15 mg / kg, about 0.85 mg / kg to about 2 mg / kg, about 1 mg / kg to about 1.85 mg / kg, about 1.15 mg / kg to about 1.7 mg / kg, about 1.3 mg / kg mg to about 1.6 mg / kg, about 1.35 mg / kg to about 1.5 mg / kg, about 2.15 mg / kg to about 3.6 mg / kg, about 2.3 mg / kg to about 3.4 mg / kg, about 2.4 mg / kg to about 3.3 mg / kg, about 2.6 mg / kg to about 3.15 mg / kg, about 2.7 mg / kg to about 3 mg / kg, about 2.8 mg / kg to about 3 mg / kg, or about 2.85 mg / kg to about 2.95 mg / kg.

[0491] In some embodiments, an effective dosage of PBLs is in the range of about 1 mg to about 500 mg, about 10 mg to about 300 mg, about 20 mg to about 250 mg, about 25 mg to about 200 mg, about 1 mg to about 50 mg, about 5 mg to about 45 mg, about 10 mg to about 40 mg, about 15 mg to about 35 mg, about 20 mg to about 30 mg, about 23 mg to about 28 mg, about 50 mg to about 150 mg, about 60 mg to about 140 mg, about 70 mg to about 130 mg, about 80 mg to about 120 mg, about 90 mg to about 110 mg, or about 95 mg to about 105 mg, about 98 mg to about 102 mg, about 150 mg to about 250 mg, about 160 mg to about 240 mg, about 170 mg to about 230 mg, about 180 mg to about 220 mg, about 190 mg to about 210 mg, about 195 mg to about 205 mg, or about 198 to about 207 mg.

[0492] An effective amount of the PBLs may be administered in either single or multiple doses by any of the accepted modes of administration of agents having similar utilities, including intranasal and transdermal routes, by intra-arterial injection, intravenously, intraperitoneally, parenterally, intramuscularly, subcutaneously, topically, by transplantation or direct injection into tumor, or by inhalation.

[0493] In some embodiments, the invention provides the pharmaceutical composition described in any of the preceding paragraphs as applicable above modified such that the pharmaceutical composition comprises 1.5×108 to 20×109 PBLs.

[0494] In some embodiments, the invention provides the pharmaceutical composition described in any of the preceding paragraphs as applicable above modified such that the pharmaceutical composition further comprises a cryopreservant.

[0495] In some embodiments, the invention provides the pharmaceutical composition described in any of the preceding paragraphs as applicable above modified such that the pharmaceutical composition further comprises at or about 5% (v / v) dimethylsulfoxide (DMSO).

[0496] In some embodiments, the invention provides the pharmaceutical composition described in any of the preceding paragraphs as applicable above modified such that the pharmaceutical composition further comprises at or about 50% (v / v) CryoStor® CS10 cryopreservation medium.

[0497] In some embodiments, the invention provides the pharmaceutical composition described in any of the preceding paragraphs as applicable above modified such that the pharmaceutical composition further comprises at or about 50% (v / v) CryoStor® CS10 cryopreservation medium and at or about 5% (v / v) DMSO.

[0498] In some embodiments, the invention provides the pharmaceutical composition described in any of the preceding paragraphs as applicable above modified such that the pharmaceutical composition further comprises a stabilizer.

[0499] In some embodiments, the invention provides the pharmaceutical composition described in any of the preceding paragraphs as applicable above modified such that the pharmaceutical composition further comprises at or about 0.5% (w / v) human serum albumin (HSA).

[0500] In some embodiments, the invention provides the pharmaceutical composition described in any of the preceding paragraphs as applicable above modified such that the pharmaceutical composition further comprises an isotonic agent.

[0501] In some embodiments, the invention provides the pharmaceutical composition described in any of the preceding paragraphs as applicable above modified such that the pharmaceutical composition further comprises at or about 50% (v / v) Plasma-Lyte A.

[0502] In some embodiments, the invention provides the pharmaceutical composition described in any of the preceding paragraphs as applicable above modified such that the pharmaceutical composition further comprises at or about 300 IU / mL of IL-2.

[0503] In some embodiments, the invention provides the pharmaceutical composition described in any of the preceding paragraphs as applicable above modified such that the pharmaceutical composition further comprises at or about 50% (v / v) CryoStor® CS10 cryopreservation medium, at or about 5% (v / v) DMSO, at or about 0.5% (w / v) human serum albumin (HSA), at or about 50% (v / v) Plasma-Lyte A, and at or about 300 IU / mL of IL-2.

[0504] In some embodiments, the invention provides the pharmaceutical composition described in any of the preceding paragraphs as applicable above modified such that the pharmaceutical composition comprises 1.5×108 to 20×109 PBLs and further comprises at or about 50% (v / v) CryoStor® CS10 cryopreservation medium, at or about 5% (v / v) DMSO, at or about 0.5% (w / v) human serum albumin (HSA), at or about 50% (v / v) Plasma-Lyte A, and at or about 300 IU / mL of IL-2.Optional Genetic Engineering of PBLs

[0505] In some embodiments, the expanded PBLs of the present invention are further manipulated before, during, or after an expansion step, including during closed, sterile manufacturing processes, each as provided herein, in order to alter protein expression in a transient manner. In some embodiments, the transiently altered protein expression is due to transient gene editing. In some embodiments, the expanded PBLs of the present invention are treated with transcription factors (TFs) and / or other molecules capable of transiently altering protein expression in the PBLs. In some embodiments, the TFs and / or other molecules that are capable of transiently altering protein expression provide for altered expression of tumor antigens and / or an alteration in the number of tumor antigen-specific T cells in a population of PBLs.

[0506] In certain embodiments, the method comprises genetically editing a population of PBLs. In certain embodiments, the method comprises genetically editing a population of PBLs provided at different stages of any of the processes described herein.

[0507] In some embodiments, the present invention includes genetic editing through nucleotide insertion, such as through ribonucleic acid (RNA) insertion, including insertion of messenger RNA (mRNA) or small (or short) interfering RNA (siRNA), into a population of PBLs for promotion of the expression of one or more proteins or inhibition of the expression of one or more proteins, as well as simultaneous combinations of both promotion of one set of proteins with inhibition of another set of proteins.

[0508] In some embodiments, the expanded PBLs of the present invention undergo transient alteration of protein expression. In some embodiments, the transient alteration of protein expression occurs at any time before, during, or after the expansion process. In some embodiments, the transient alteration of protein expression occurs at any step within the expansion process. In some embodiments, the transient alteration of protein expression occurs in the bulk PBL population prior to a first expansion. In some embodiments, the transient alteration of protein expression occurs during the first expansion. In some embodiments, the transient alteration of protein expression occurs after the first expansion, including, for example in the PBL population in transition between the first and second expansion (e.g. the second population of PBLs as described herein. In some embodiments, the transient alteration of protein expression occurs in the bulk PBL population prior to second expansion. In some embodiments, the transient alteration of protein expression occurs during the second expansion, including, for example in the PBL population being expanded (e.g. the third population of PBLs). In some embodiments, the transient alteration of protein expression occurs after the second expansion.

[0509] In an embodiment, a method of transiently altering protein expression in a population of PBLs includes the step of electroporation. Electroporation methods are known in the art and are described, e.g., in Tsong, Biophys. J. 1991, 60, 297-306, and U.S. Patent Application Publication No. 2014 / 0227237 A1, the disclosures of each of which are incorporated by reference herein. In an embodiment, a method of transiently altering protein expression in population of PBLs includes the step of calcium phosphate transfection. Calcium phosphate transfection methods (calcium phosphate DNA precipitation, cell surface coating, and endocytosis) are known in the art and are described in Graham and van der Eb, Virology 1973, 52, 456-467; Wigler, et al., Proc. Natl. Acad. Sci. 1979, 76, 1373-1376; and Chen and Okayarea, Mol. Cell. Biol. 1987, 7, 2745-2752; and in U.S. Pat. No. 5,593,875, the disclosures of each of which are incorporated by reference herein. In an embodiment, a method of transiently altering protein expression in a population of PBLs includes the step of liposomal transfection. Liposomal transfection methods, such as methods that employ a 1:1 (w / w) liposome formulation of the cationic lipid N-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammonium chloride (DOTMA) and dioleoyl phophotidylethanolamine (DOPE) in filtered water, are known in the art and are described in Rose, et al., Biotechniques 1991, 10, 520-525 and Felgner, et al., Proc. Natl. Acad. Sci. USA, 1987, 84, 7413-7417 and in U.S. Pat. Nos. 5,279,833; 5,908,635; 6,056,938; 6,110,490; 6,534,484; and 7,687,070, the disclosures of each of which are incorporated by reference herein. In an embodiment, a method of transiently altering protein expression in a population of PBLs includes the step of transfection using methods described in U.S. Pat. Nos. 5,766,902; 6,025,337; 6,410,517; 6,475,994; and 7,189,705; the disclosures of each of which are incorporated by reference herein.

[0510] In some embodiments, transient alteration of protein expression results in an increase in Stem Memory T cells (TSCMs). TSCMs are early progenitors of antigen-experienced central memory T cells. TSCMs generally display the long-term survival, self-renewal, and multipotency abilities that define stem cells, and are generally desirable for the generation of effective TIL products. TSCM have shown enhanced anti-tumor activity compared with other T cell subsets in mouse models of adoptive cell transfer (Gattinoni et al. Nat Med 2009, 2011; Gattinoni, Nature Rev. Cancer, 2012; Cieri et al. Blood 2013). In some embodiments, transient alteration of protein expression results in a TIL population with a composition comprising a high proportion of TSCM. In some embodiments, transient alteration of protein expression results in an at least 5%, at least 10%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% increase in TSCM percentage. In some embodiments, transient alteration of protein expression results in an at least a 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, or 10-fold increase in TSCMs in the TIL population. In some embodiments, transient alteration of protein expression results in a TIL population with at least at least 5%, at least 10%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% TSCMs. In some embodiments, transient alteration of protein expression results in a therapeutic TIL population with at least at least 5%, at least 10%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% TSCMs.

[0511] In some embodiments, transient alteration of protein expression results in rejuvenation of antigen-experienced T-cells. In some embodiments, rejuvenation includes, for example, increased proliferation, increased T-cell activation, and / or increased antigen recognition.

[0512] In some embodiments, transient alteration of protein expression alters the expression in a large fraction of the T-cells in order to preserve the tumor-derived TCR repertoire. In some embodiments, transient alteration of protein expression does not alter the tumor-derived TCR repertoire. In some embodiments, transient alteration of protein expression maintains the tumor-derived TCR repertoire.

[0513] In some embodiments, transient alteration of protein results in altered expression of a particular gene. In some embodiments, the transient alteration of protein expression targets a gene including but not limited to PD-1 (also referred to as PDCD1 or CC279), TGFBR2, CCR4 / 5, CBLB (CBL-B), CISH, CCRs (chimeric co-stimulatory receptors), IL-2, IL-12, IL-15, IL-21, NOTCH 1 / 2 ICD, TIM3, LAG3, TIGIT, TGFβ, CCR2, CCR4, CCR5, CXCR1, CXCR2, CSCR3, CCL2 (MCP-1), CCL3 (MIP-1α), CCL4 (MIP1-β), CCL5 (RANTES), CXCL1 / CXCL8, CCL22, CCL17, CXCL1 / CXCL8, VHL, CD44, PIK3CD, SOCS1, and / or cAMP protein kinase A (PKA). In some embodiments, the transient alteration of protein expression targets a gene selected from the group consisting of PD-1, TGFBR2, CCR4 / 5, CBLB (CBL-B), CISH, CCRs (chimeric co-stimulatory receptors), IL-2, IL-12, IL-15, IL-21, NOTCH 1 / 2 ICD, TIM3, LAG3, TIGIT, TGFβ, CCR2, CCR4, CCR5, CXCR1, CXCR2, CSCR3, CCL2 (MCP-1), CCL3 (MIP-1α), CCL4 (MIP1-β), CCL5 (RANTES), CXCL1 / CXCL8, CCL22, CCL17, CXCL1 / CXCL8, VHL, CD44, PIK3CD, SOCS1, and / or cAMP protein kinase A (PKA). In some embodiments, the transient alteration of protein expression targets PD-1. In some embodiments, the transient alteration of protein expression targets TGFBR2. In some embodiments, the transient alteration of protein expression targets CCR4 / 5. In some embodiments, the transient alteration of protein expression targets CBLB. In some embodiments, the transient alteration of protein expression targets CISH. In some embodiments, the transient alteration of protein expression targets CCRs (chimeric co-stimulatory receptors). In some embodiments, the transient alteration of protein expression targets IL-2. In some embodiments, the transient alteration of protein expression targets IL-12. In some embodiments, the transient alteration of protein expression targets IL-15. In some embodiments, the transient alteration of protein expression targets IL-21. In some embodiments, the transient alteration of protein expression targets NOTCH 1 / 2 ICD. In some embodiments, the transient alteration of protein expression targets TIM3. In some embodiments, the transient alteration of protein expression targets LAG3. In some embodiments, the transient alteration of protein expression targets TIGIT. In some embodiments, the transient alteration of protein expression targets TGFβ. In some embodiments, the transient alteration of protein expression targets CCR1. In some embodiments, the transient alteration of protein expression targets CCR2. In some embodiments, the transient alteration of protein expression targets CCR4. In some embodiments, the transient alteration of protein expression targets CCR5. In some embodiments, the transient alteration of protein expression targets CXCR1. In some embodiments, the transient alteration of protein expression targets CXCR2. In some embodiments, the transient alteration of protein expression targets CSCR3. In some embodiments, the transient alteration of protein expression targets CCL2 (MCP-1). In some embodiments, the transient alteration of protein expression targets CCL3 (MIP-1α). In some embodiments, the transient alteration of protein expression targets CCL4 (MIP1-β). In some embodiments, the transient alteration of protein expression targets CCL5 (RANTES). In some embodiments, the transient alteration of protein expression targets CXCL1. In some embodiments, the transient alteration of protein expression targets CXCL8. In some embodiments, the transient alteration of protein expression targets CCL22. In some embodiments, the transient alteration of protein expression targets CCL17. In some embodiments, the transient alteration of protein expression targets VHL. In some embodiments, the transient alteration of protein expression targets CD44. In some embodiments, the transient alteration of protein expression targets PIK3CD. In some embodiments, the transient alteration of protein expression targets SOCS1. In some embodiments, the transient alteration of protein expression targets cAMP protein kinase A (PKA).

[0514] In some embodiments, the transient alteration of protein expression results in increased and / or overexpression of a chemokine receptor. In some embodiments, the chemokine receptor that is overexpressed by transient protein expression includes a receptor with a ligand that includes but is not limited to CCL2 (MCP-1), CCL3 (MIP-1α), CCL4 (MIP1-β), CCL5 (RANTES), CXCL1, CXCL8, CCL22, and / or CCL17.

[0515] In some embodiments, the transient alteration of protein expression results in a decrease and / or reduced expression of PD-1, CTLA-4, TIM-3, LAG-3, TIGIT, TGFβR2, and / or TGFβ (including resulting in, for example, TGFβ pathway blockade). In some embodiments, the transient alteration of protein expression results in a decrease and / or reduced expression of CBLB (CBL-B). In some embodiments, the transient alteration of protein expression results in a decrease and / or reduced expression of CISH.

[0516] In some embodiments, the transient alteration of protein expression results in increased and / or overexpression of chemokine receptors in order to, for example, improve TIL trafficking or movement to the tumor site. In some embodiments, the transient alteration of protein expression results in increased and / or overexpression of a CCR (chimeric co-stimulatory receptor). In some embodiments, the transient alteration of protein expression results in increased and / or overexpression of a chemokine receptor selected from the group consisting of CCR1, CCR2, CCR4, CCR5, CXCR1, CXCR2, and / or CSCR3.

[0517] In some embodiments, the transient alteration of protein expression results in increased and / or overexpression of an interleukin. In some embodiments, the transient alteration of protein expression results in increased and / or overexpression of an interleukin selected from the group consisting of IL-2, IL-12, IL-15, and / or IL-21.

[0518] In some embodiments, the transient alteration of protein expression results in increased and / or overexpression of NOTCH 1 / 2 ICD. In some embodiments, the transient alteration of protein expression results in increased and / or overexpression of VHL. In some embodiments, the transient alteration of protein expression results in increased and / or overexpression of CD44. In some embodiments, the transient alteration of protein expression results in increased and / or overexpression of PIK3CD. In some embodiments, the transient alteration of protein expression results in increased and / or overexpression of SOCS1,

[0519] In some embodiments, the transient alteration of protein expression results in decreased and / or reduced expression of cAMP protein kinase A (PKA).

[0520] In some embodiments, the transient alteration of protein expression results in decreased and / or reduced expression of a molecule selected from the group consisting of PD-1, LAG3, TIM3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBLB, BAFF (BR3), and combinations thereof. In some embodiments, the transient alteration of protein expression results in decreased and / or reduced expression of two molecules selected from the group consisting of PD-1, LAG3, TIM3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBLB, BAFF (BR3), and combinations thereof. In some embodiments, the transient alteration of protein expression results in decreased and / or reduced expression of PD-1 and one molecule selected from the group consisting of LAG3, TIM3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBLB, BAFF (BR3), and combinations thereof. In some embodiments, the transient alteration of protein expression results in decreased and / or reduced expression of PD-1, LAG-3, CISH, CBLB, TIM3, and combinations thereof. In some embodiments, the transient alteration of protein expression results in decreased and / or reduced expression of PD-1 and one of LAG3, CISH, CBLB, TIM3, and combinations thereof. In some embodiments, the transient alteration of protein expression results in decreased and / or reduced expression of PD-1 and LAG3. In some embodiments, the transient alteration of protein expression results in decreased and / or reduced expression of PD-1 and CISH. In some embodiments, the transient alteration of protein expression results in decreased and / or reduced expression of PD-1 and CBLB. In some embodiments, the transient alteration of protein expression results in decreased and / or reduced expression of LAG3 and CISH. In some embodiments, the transient alteration of protein expression results in decreased and / or reduced expression of LAG3 and CBLB. In some embodiments, the transient alteration of protein expression results in decreased and / or reduced expression of CISH and CBLB. In some embodiments, the transient alteration of protein expression results in decreased and / or reduced expression of TIM3 and PD-1. In some embodiments, the transient alteration of protein expression results in decreased and / or reduced expression of TIM3 and LAG3. In some embodiments, the transient alteration of protein expression results in decreased and / or reduced expression of TIM3 and CISH. In some embodiments, the transient alteration of protein expression results in decreased and / or reduced expression of TIM3 and CBLB.

[0521] In some embodiments, an adhesion molecule selected from the group consisting of CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, and combinations thereof, is inserted by a gammaretroviral or lentiviral method into the first population of PBLs, second population of PBLs, or harvested population of PBLs (e.g., the expression of the adhesion molecule is increased).

[0522] In some embodiments, the transient alteration of protein expression results in decreased and / or reduced expression of a molecule selected from the group consisting of PD-1, LAG3, TIM3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBLB, BAFF (BR3), and combinations thereof, and increased and / or enhanced expression of CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, and combinations thereof. In some embodiments, the transient alteration of protein expression results in decreased and / or reduced expression of a molecule selected from the group consisting of PD-1, LAG3, TIM3, CISH, CBLB, and combinations thereof, and increased and / or enhanced expression of CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, and combinations thereof.

[0523] In some embodiments, there is a reduction in expression of about 5%, about 10%, about 10%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, there is a reduction in expression of at least about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, there is a reduction in expression of at least about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, there is a reduction in expression of at least about 80%, about 85%, about 90%, or about 95%. In some embodiments, there is a reduction in expression of at least about 85%, about 90%, or about 95%. In some embodiments, there is a reduction in expression of at least about 80%. In some embodiments, there is a reduction in expression of at least about 85%, In some embodiments, there is a reduction in expression of at least about 90%. In some embodiments, there is a reduction in expression of at least about 95%. In some embodiments, there is a reduction in expression of at least about 99%.

[0524] In some embodiments, there is an increase in expression of about 5%, about 10%, about 10%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, there is an increase in expression of at least about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, there is an increase in expression of at least about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, there is an increase in expression of at least about 80%, about 85%, about 90%, or about 95%. In some embodiments, there is an increase in expression of at least about 85%, about 90%, or about 95%. In some embodiments, there is an increase in expression of at least about 80%. In some embodiments, there is an increase in expression of at least about 85%, In some embodiments, there is an increase in expression of at least about 90%. In some embodiments, there is an increase in expression of at least about 95%. In some embodiments, there is an increase in expression of at least about 99%.

[0525] In some embodiments, transient alteration of protein expression is induced by treatment of the PBLs with transcription factors (TFs) and / or other molecules capable of transiently altering protein expression in the PBLs. In some embodiments, the SQZ vector-free microfluidic platform is employed for intracellular delivery of the transcription factors (TFs) and / or other molecules capable of transiently altering protein expression. Such methods demonstrating the ability to deliver proteins, including transcription factors, to a variety of primary human cells, including T cells (Sharei et al. PNAS 2013, as well as Sharei et al. PLOS ONE 2015 and Greisbeck et al. J. Immunology vol. 195, 2015) have been described; see, for example, International Patent Publications WO 2013 / 059343A1, WO 2017 / 008063A1, and WO 2017 / 123663A1, all of which are incorporated by reference herein in their entireties. Such methods as described in International Patent Publications WO 2013 / 059343A1, WO 2017 / 008063A1, and WO 2017 / 123663A1 can be employed with the present invention in order to expose a population of PBLs to transcription factors (TFs) and / or other molecules capable of inducing transient protein expression, wherein said TFs and / or other molecules capable of inducing transient protein expression provide for increased expression of tumor antigens and / or an increase in the number of tumor antigen-specific T cells in the population of PBLs, thus resulting in reprogramming of the TIL population and an increase in therapeutic efficacy of the reprogrammed TIL population as compared to a non-reprogrammed TIL population. In some embodiments, the reprogramming results in an increased subpopulation of effector T cells and / or central memory T cells relative to the starting or prior population (i.e., prior to reprogramming) population of PBLs, as described herein.

[0526] In some embodiments, the transcription factor (TF) includes but is not limited to TCF-1, NOTCH 1 / 2 ICD, and / or MYB. In some embodiments, the transcription factor (TF) is TCF-1. In some embodiments, the transcription factor (TF) is NOTCH 1 / 2 ICD. In some embodiments, the transcription factor (TF) is MYB. In some embodiments, the transcription factor (TF) is administered with induced pluripotent stem cell culture (iPSC), such as the commercially available KNOCKOUT Serum Replacement (Gibco / ThermoFisher), to induce additional TIL reprogramming. In some embodiments, the transcription factor (TF) is administered with an iPSC cocktail to induce additional TIL reprogramming. In some embodiments, the transcription factor (TF) is administered without an iPSC cocktail. In some embodiments, reprogramming results in an increase in the percentage of TSCMs. In some embodiments, reprogramming results in an increase in the percentage of TSCMs by about 5%, about 10%, about 10%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% TSCMs.

[0527] In some embodiments, a method of transient altering protein expression, as described above, may be combined with a method of genetically modifying a population of PBLs by including the step of stable incorporation of genes for production of one or more proteins. In certain embodiments, the method comprises a step of genetically modifying a population of PBLs. In certain embodiments, the method comprises genetically modifying the first population of PBLs, the second population of PBLs and / or the third population of PBLs. In an embodiment, a method of genetically modifying a population of PBLs includes the step of retroviral transduction. In an embodiment, a method of genetically modifying a population of PBLs includes the step of lentiviral transduction. Lentiviral transduction systems are known in the art and are described, e.g., in Levine, et al., Proc. Nat'l Acad. Sci. 2006, 103, 17372-77; Zufferey, et al., Nat. Biotechnol. 1997, 15, 871-75; Dull, et al., J Virology 1998, 72, 8463-71, and U.S. Pat. No. 6,627,442, the disclosures of each of which are incorporated by reference herein. In an embodiment, a method of genetically modifying a population of PBLs includes the step of gamma-retroviral transduction. Gamma-retroviral transduction systems are known in the art and are described, e.g., Cepko and Pear, Cur. Prot. Mol. Biol. 1996, 9.9.1-9.9.16, the disclosure of which is incorporated by reference herein. In an embodiment, a method of genetically modifying a population of PBLs includes the step of transposon-mediated gene transfer. Transposon-mediated gene transfer systems are known in the art and include systems wherein the transposase is provided as DNA expression vector or as an expressible RNA or a protein such that long-term expression of the transposase does not occur in the transgenic cells, for example, a transposase provided as an mRNA (e.g., an mRNA comprising a cap and poly-A tail). Suitable transposon-mediated gene transfer systems, including the salmonid-type Tel-like transposase (SB or Sleeping Beauty transposase), such as SB10, SB11, and SB100x, and engineered enzymes with increased enzymatic activity, are described in, e.g., Hackett, et al., Mol. Therapy 2010, 18, 674-83 and U.S. Pat. No. 6,489,458, the disclosures of each of which are incorporated by reference herein.

[0528] In some embodiments, transient alteration of protein expression is a reduction in expression induced by self-delivering RNA interference (sdRNA), which is a chemically-synthesized asymmetric siRNA duplex with a high percentage of 2′-OH substitutions (typically fluorine or —OCH3) which comprises a 20-nucleotide antisense (guide) strand and a 13 to 15 base sense (passenger) strand conjugated to cholesterol at its 3′ end using a tetraethylenglycol (TEG) linker. In some embodiments, the method comprises transient alteration of protein expression in a population of PBLs, comprising the use of self-delivering RNA interference (sdRNA), which is a chemically-synthesized asymmetric siRNA duplex with a high percentage of 2′-OH substitutions (typically fluorine or —OCH3) which comprises a 20-nucleotide antisense (guide) strand and a 13 to 15 base sense (passenger) strand conjugated to cholesterol at its 3′ end using a tetraethylenglycol (TEG) linker. Methods of using sdRNA have been described in Khvorova and Watts, Nat. Biotechnol. 2017, 35, 238-248; Byrne, et al., J. Ocul. Pharmacol. Ther. 2013, 29, 855-864; and Ligtenberg, et al., Mol. Therapy, 2018, 26, 1482-1493, the disclosures of which are incorporated by reference herein. In an embodiment, delivery of sdRNA to a TIL population is accomplished without use of electroporation, SQZ, or other methods, instead using a 1 to 3 day period in which a TIL population is exposed to sdRNA at a concentration of 1 μM / 10,000 PBLs in medium. In certain embodiments, the method comprises delivery sdRNA to a PBLs population comprising exposing the PBLs population to sdRNA at a concentration of 1 μM / 10,000 PBLs in medium for a period of between 1 to 3 days. In an embodiment, delivery of sdRNA to a TIL population is accomplished using a 1 to 3 day period in which a TIL population is exposed to sdRNA at a concentration of 10 μM / 10,000 PBLs in medium. In an embodiment, delivery of sdRNA to a TIL population is accomplished using a 1 to 3 day period in which a TIL population is exposed to sdRNA at a concentration of 50 μM / 10,000 PBLs in medium. In an embodiment, delivery of sdRNA to a TIL population is accomplished using a 1 to 3 day period in which a TIL population is exposed to sdRNA at a concentration of between 0.1 μm / 10,000 PBLs and 50 μM / 10,000 PBLs in medium. In an embodiment, delivery of sdRNA to a TIL population is accomplished using a 1 to 3 day period in which a TIL population is exposed to sdRNA at a concentration of between 0.1 μM / 10,000 PBLs and 50 μM / 10,000 PBLs in medium, wherein the exposure to sdRNA is performed two, three, four, or five times by addition of fresh sdRNA to the media. Other suitable processes are described, for example, in U.S. Patent Application Publication No. US 2011 / 0039914 A1, US 2013 / 0131141 A1, and US 2013 / 0131142 A1, and U.S. Pat. No. 9,080,171, the disclosures of which are incorporated by reference herein.

[0529] In some embodiments, sdRNA is inserted into a population of PBLs during manufacturing. In some embodiments, the sdRNA encodes RNA that interferes with NOTCH 1 / 2 ICD, PD-1, CTLA-4 TIM-3, LAG-3, TIGIT, TGFβ, TGFBR2, cAMP protein kinase A (PKA), BAFF BR3, CISH, and / or CBLB. In some embodiments, the reduction in expression is determined based on a percentage of gene silencing, for example, as assessed by flow cytometry and / or qPCR. In some embodiments, there is a reduction in expression of about 5%, about 10%, about 10%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, there is a reduction in expression of at least about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, there is a reduction in expression of at least about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, there is a reduction in expression of at least about 80%, about 85%, about 90%, or about 95%. In some embodiments, there is a reduction in expression of at least about 85%, about 90%, or about 95%. In some embodiments, there is a reduction in expression of at least about 80%. In some embodiments, there is a reduction in expression of at least about 85%. In some embodiments, there is a reduction in expression of at least about 90%. In some embodiments, there is a reduction in expression of at least about 95%. In some embodiments, there is a reduction in expression of at least about 99%.

[0530] The self-deliverable RNAi technology based on the chemical modification of siRNAs can be employed with the methods of the present invention to successfully deliver the sdRNAs to the PBLs as described herein. The combination of backbone modifications with asymmetric siRNA structure and a hydrophobic ligand (see, for example, Ligtenberg, et al., Mol. Therapy, 2018 and US20160304873) allow sdRNAs to penetrate cultured mammalian cells without additional formulations and methods by simple addition to the culture media, capitalizing on the nuclease stability of sdRNAs. This stability allows the support of constant levels of RNAi-mediated reduction of target gene activity simply by maintaining the active concentration of sdRNA in the media. While not being bound by theory, the backbone stabilization of sdRNA provides for extended reduction in gene expression effects which can last for months in non-dividing cells.

[0531] In some embodiments, over 95% transfection efficiency of PBLs and a reduction in expression of the target by various specific sdRNA occurs. In some embodiments, sdRNAs containing several unmodified ribose residues were replaced with fully modified sequences to increase potency and / or the longevity of RNAi effect. In some embodiments, a reduction in expression effect is maintained for 12 hours, 24 hours, 36 hours, 48 hours, 5 days, 6 days, 7 days, or 8 days or more. In some embodiments, the reduction in expression effect decreases at 10 days or more post sdRNA treatment of the PBLs. In some embodiments, more than 70% reduction in expression of the target expression is maintained. In some embodiments, more than 70% reduction in expression of the target expression is maintained in PBLs. In some embodiments, a reduction in expression in the PD-1 / PD-L1 pathway allows for the PBLs to exhibit a more potent in vivo effect, which is in some embodiments, due to the avoidance of the suppressive effects of the PD-1 / PD-L1 pathway. In some embodiments, a reduction in expression of PD-1 by sdRNA results in an increase TIL proliferation.

[0532] Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, is a double stranded RNA molecule, generally 19-25 base pairs in length. siRNA is used in RNA interference (RNAi), where it interferes with expression of specific genes with complementary nucleotide sequences.

[0533] Double stranded DNA (dsRNA) can be generally used to define any molecule comprising a pair of complementary strands of RNA, generally a sense (passenger) and antisense (guide) strands, and may include single-stranded overhang regions. The term dsRNA, contrasted with siRNA, generally refers to a precursor molecule that includes the sequence of an siRNA molecule which is released from the larger dsRNA molecule by the action of cleavage enzyme systems, including Dicer.

[0534] sdRNA (self-deliverable RNA) are a new class of covalently modified RNAi compounds that do not require a delivery vehicle to enter cells and have improved pharmacology compared to traditional siRNAs. “Self-deliverable RNA” or “sdRNA” is a hydrophobically modified RNA interfering-antisense hybrid, demonstrated to be highly efficacious in vitro in primary cells and in vivo upon local administration. Robust uptake and / or silencing without toxicity has been demonstrated. sdRNAs are generally asymmetric chemically modified nucleic acid molecules with minimal double stranded regions. sdRNA molecules typically contain single stranded regions and double stranded regions, and can contain a variety of chemical modifications within both the single stranded and double stranded regions of the molecule. Additionally, the sdRNA molecules can be attached to a hydrophobic conjugate such as a conventional and advanced sterol-type molecule, as described herein. sdRNAs and associated methods for making such sdRNAs have also been described extensively in, for example, US20160304873, WO2010033246, WO2017070151, WO2009102427, WO2011119887, WO2010033247A2, WO2009045457, WO2011119852, all of which are incorporated by reference herein in their entireties for all purposes. To optimize sdRNA structure, chemistry, targeting position, sequence preferences, and the like, a proprietary algorithm has been developed and utilized for sdRNA potency prediction (see, for example, US 20160304873). Based on these analyses, functional sdRNA sequences have been generally defined as having over 70% reduction in expression at 1 μM concentration, with a probability over 40%.

[0535] In some embodiments, the sdRNA sequences used in the invention exhibit a 70% reduction in expression of the target gene. In some embodiments, the sdRNA sequences used in the invention exhibit a 75% reduction in expression of the target gene.

[0536] In some embodiments, the sdRNA sequences used in the invention exhibit an 80% reduction in expression of the target gene. In some embodiments, the sdRNA sequences used in the invention exhibit an 85% reduction in expression of the target gene. In some embodiments, the sdRNA sequences used in the invention exhibit a 90% reduction in expression of the target gene. In some embodiments, the sdRNA sequences used in the invention exhibit a 95% reduction in expression of the target gene. In some embodiments, the sdRNA sequences used in the invention exhibit a 99% reduction in expression of the target gene. In some embodiments, the sdRNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 0.25 μM to about 4 μM. In some embodiments, the sdRNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 0.25 μM. In some embodiments, the sdRNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 0.5 μM. In some embodiments, the sdRNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 0.75 μM. In some embodiments, the sdRNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 1.0 μM. In some embodiments, the sdRNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 1.25 μM. In some embodiments, the sdRNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 1.5 μM. In some embodiments, the sdRNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 1.75 μM. In some embodiments, the sdRNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 2.0 μM. In some embodiments, the sdRNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 2.25 μM. In some embodiments, the sdRNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 2.5 μM. In some embodiments, the sdRNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 2.75 μM. In some embodiments, the sdRNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 3.0 μM. In some embodiments, the sdRNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 3.25 μM. In some embodiments, the sdRNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 3.5 μM. In some embodiments, the sdRNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 3.75 μM. In some embodiments, the sdRNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 4.0 μM.

[0537] In some embodiments, the oligonucleotide agents comprise one or more modification to increase stability and / or effectiveness of the therapeutic agent, and to effect efficient delivery of the oligonucleotide to the cells or tissue to be treated. Such modifications can include a 2′-O-methyl modification, a 2′-O-Fluro modification, a diphosphorothioate modification, 2′ F modified nucleotide, a2′-O-methyl modified and / or a 2′deoxy nucleotide. In some embodiments, the oligonucleotide is modified to include one or more hydrophobic modifications including, for example, sterol, cholesterol, vitamin D, naphtyl, isobutyl, benzyl, indol, tryptophane, and / or phenyl. In an additional particular embodiment, chemically modified nucleotides are combination of phosphorothioates, 2′-O-methyl, 2′deoxy, hydrophobic modifications and phosphorothioates. In some embodiments, the sugars can be modified and modified sugars can include but are not limited to D-ribose, 2′-O-alkyl (including 2′-O-methyl and 2′-O-ethyl), i.e., 2′-alkoxy, 2′-amino, 2′-S-alkyl, 2′-halo (including 2′-fluoro), T-methoxyethoxy, 2′-allyloxy (—OCH2CH═CH2), 2′-propargyl, 2′-propyl, ethynyl, ethenyl, propenyl, and cyano and the like. In one embodiment, the sugar moiety can be a hexose and incorporated into an oligonucleotide as described (Augustyns, K., et al., Nucl. Acids. Res. 18:4711 (1992)).

[0538] In some embodiments, the double-stranded oligonucleotide of the invention is double-stranded over its entire length, i.e., with no overhanging single-stranded sequence at either end of the molecule, i.e., is blunt-ended. In some embodiments, the individual nucleic acid molecules can be of different lengths. In other words, a double-stranded oligonucleotide of the invention is not double-stranded over its entire length. For instance, when two separate nucleic acid molecules are used, one of the molecules, e.g., the first molecule comprising an antisense sequence, can be longer than the second molecule hybridizing thereto (leaving a portion of the molecule single-stranded). In some embodiments, when a single nucleic acid molecule is used a portion of the molecule at either end can remain single-stranded.

[0539] In some embodiments, a double-stranded oligonucleotide of the invention contains mismatches and / or loops or bulges, but is double-stranded over at least about 70% of the length of the oligonucleotide. In some embodiments, a double-stranded oligonucleotide of the invention is double-stranded over at least about 80% of the length of the oligonucleotide. In another embodiment, a double-stranded oligonucleotide of the invention is double-stranded over at least about 90%-95% of the length of the oligonucleotide. In some embodiments, a double-stranded oligonucleotide of the invention is double-stranded over at least about 96%-98% of the length of the oligonucleotide. In some embodiments, the double-stranded oligonucleotide of the invention contains at least or up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mismatches.

[0540] In some embodiments, the oligonucleotide can be substantially protected from nucleases e.g., by modifying the 3′ or 5′ linkages (e.g., U.S. Pat. No. 5,849,902 and WO 98 / 13526). For example, oligonucleotides can be made resistant by the inclusion of a “blocking group.” The term “blocking group” as used herein refers to substituents (e.g., other than OH groups) that can be attached to oligonucleotides or nucleomonomers, either as protecting groups or coupling groups for synthesis (e.g., FITC, propyl (CH2—CH2—CH3), glycol (—O—CH2—CH2—O—) phosphate (PO32″), hydrogen phosphonate, or phosphoramidite). “Blocking groups” can also include “end blocking groups” or “exonuclease blocking groups” which protect the 5′ and 3′ termini of the oligonucleotide, including modified nucleotides and non-nucleotide exonuclease resistant structures.

[0541] In some embodiments, at least a portion of the contiguous polynucleotides within the sdRNA are linked by a substitute linkage, e.g., a phosphorothioate linkage.

[0542] In some embodiments, chemical modification can lead to at least a 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 enhancements in cellular uptake. In some embodiments, at least one of the C or U residues includes a hydrophobic modification. In some embodiments, a plurality of Cs and Us contain a hydrophobic modification. In some embodiments, at least 10%, 15%, 20%, 30%, 40%, 50%, 55%, 60% 65%, 70%, 75%, 80%, 85%, 90% or at least 95% of the Cs and Us can contain a hydrophobic modification. In some embodiments, all of the Cs and Us contain a hydrophobic modification.

[0543] In some embodiments, the sdRNA or sd-rxRNAs exhibit enhanced endosomal release of sd-rxRNA molecules through the incorporation of protonatable amines. In some embodiments, protonatable amines are incorporated in the sense strand (in the part of the molecule which is discarded after RISC loading). In some embodiments, the sdRNA compounds of the invention comprise an asymmetric compound comprising a duplex region (required for efficient RISC entry of 10-15 bases long) and single stranded region of 4-12 nucleotides long; with a 13 nucleotide duplex. In some embodiments, a 6 nucleotide single stranded region is employed. In some embodiments, the single stranded region of the sdRNA comprises 2-12 phosphorothioate intemucleotide linkages (referred to as phosphorothioate modifications). In some embodiments, 6-8 phosphorothioate intemucleotide linkages are employed. In some embodiments, the sdRNA compounds of the invention also include a unique chemical modification pattern, which provides stability and is compatible with RISC entry.

[0544] The guide strand, for example, may also be modified by any chemical modification which confirms stability without interfering with RISC entry. In some embodiments, the chemical modification pattern in the guide strand includes the majority of C and U nucleotides being 2′ F modified and the 5′ end being phosphorylated.

[0545] In some embodiments, at least 30% of the nucleotides in the sdRNA or sd-rxRNA are modified. In some embodiments, at least 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79% 80% 81%, 82%, 83%, 84%, 85% 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the nucleotides in the sdRNA or sd-rxRNA are modified. In some embodiments, 100% of the nucleotides in the sdRNA or sd-rxRNA are modified.

[0546] In some embodiments, the sdRNA molecules have minimal double stranded regions. In some embodiments the region of the molecule that is double stranded ranges from 8-15 nucleotides long. In some embodiments, the region of the molecule that is double stranded is 8, 9, 10, 11, 12, 13, 14 or 15 nucleotides long. In some embodiments the double stranded region is 13 nucleotides long. There can be 100% complementarity between the guide and passenger strands, or there may be one or more mismatches between the guide and passenger strands. In some embodiments, on one end of the double stranded molecule, the molecule is either blunt-ended or has a one-nucleotide overhang. The single stranded region of the molecule is in some embodiments between 4-12 nucleotides long. In some embodiments, the single stranded region can be 4, 5, 6, 7, 8, 9, 10, 11 or 12 nucleotides long. In some embodiments, the single stranded region can also be less than 4 or greater than 12 nucleotides long. In certain embodiments, the single stranded region is 6 or 7 nucleotides long.

[0547] In some embodiments, the sdRNA molecules have increased stability. In some instances, a chemically modified sdRNA or sd-rxRNA molecule has a half-life in media that is longer than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more than 24 hours, including any intermediate values. In some embodiments, the sd-rxRNA has a half-life in media that is longer than 12 hours.

[0548] In some embodiments, the sdRNA is optimized for increased potency and / or reduced toxicity. In some embodiments, nucleotide length of the guide and / or passenger strand, and / or the number of phosphorothioate modifications in the guide and / or passenger strand, can in some aspects influence potency of the RNA molecule, while replacing 2′-fluoro (2′F) modifications with 2′-O-methyl (2′OMe) modifications can in some aspects influence toxicity of the molecule. In some embodiments, reduction in 2′F content of a molecule is predicted to reduce toxicity of the molecule. In some embodiments, the number of phosphorothioate modifications in an RNA molecule can influence the uptake of the molecule into a cell, for example the efficiency of passive uptake of the molecule into a cell. In some embodiments, the sdRNA has no 2′F modification and yet are characterized by equal efficacy in cellular uptake and tissue penetration.

[0549] In some embodiments, a guide strand is approximately 18-19 nucleotides in length and has approximately 2-14 phosphate modifications. For example, a guide strand can contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more than 14 nucleotides that are phosphate-modified. The guide strand may contain one or more modifications that confer increased stability without interfering with RISC entry. The phosphate modified nucleotides, such as phosphorothioate modified nucleotides, can be at the 3′ end, 5′ end or spread throughout the guide strand. In some embodiments, the 3′ terminal 10 nucleotides of the guide strand contain 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 phosphorothioate modified nucleotides. The guide strand can also contain 2′F and / or 2′OMe modifications, which can be located throughout the molecule. In some embodiments, the nucleotide in position one of the guide strand (the nucleotide in the most 5′ position of the guide strand) is 2′OMe modified and / or phosphorylated. C and U nucleotides within the guide strand can be 2′F modified. For example, C and U nucleotides in positions 2-10 of a 19 nt guide strand (or corresponding positions in a guide strand of a different length) can be 2′F modified. C and U nucleotides within the guide strand can also be 2′OMe modified. For example, C and U nucleotides in positions 11-18 of a 19 nt guide strand (or corresponding positions in a guide strand of a different length) can be 2′OMe modified. In some embodiments, the nucleotide at the most 3′ end of the guide strand is unmodified. In certain embodiments, the majority of Cs and Us within the guide strand are 2′F modified and the 5′ end of the guide strand is phosphorylated. In other embodiments, position 1 and the Cs or Us in positions 11-18 are 2′OMe modified and the 5′ end of the guide strand is phosphorylated. In other embodiments, position 1 and the Cs or Us in positions 11-18 are 2′OMe modified, the 5′ end of the guide strand is phosphorylated, and the Cs or Us in position 2-10 are 2′F modified.

[0550] The self-deliverable RNAi technology provides a method of directly transfecting cells with the RNAi agent, without the need for additional formulations or techniques. The ability to transfect hard-to-transfect cell lines, high in vivo activity, and simplicity of use, are characteristics of the compositions and methods that present significant functional advantages over traditional siRNA-based techniques, and as such, the sdRNA methods are employed in several embodiments related to the methods of reduction in expression of the target gene in the PBLs of the present invention. The sdRNAi methods allow direct delivery of chemically synthesized compounds to a wide range of primary cells and tissues, both ex-vivo and in vivo. The sdRNAs described in some embodiments of the invention herein are commercially available from Advirna LLC, Worcester, MA, USA.

[0551] The sdRNA are formed as hydrophobically-modified siRNA-antisense oligonucleotide hybrid structures, and are disclosed, for example in Byrne et al., December 2013, J. Ocular Pharmacology and Therapeutics, 29(10): 855-864, incorporated by reference herein in its entirety.

[0552] In some embodiments, the sdRNA oligonucleotides can be delivered to the PBLs described herein using sterile electroporation. In certain embodiments, the method comprises sterile electroporation of a population of PBLs to deliver sdRNA oligonucleotides.

[0553] In some embodiments, the oligonucleotides can be delivered to the cells in combination with a transmembrane delivery system. In some embodiments, this transmembrane delivery system comprises lipids, viral vectors, and the like. In some embodiments, the oligonucleotide agent is a self-delivery RNAi agent, that does not require any delivery agents. In certain embodiments, the method comprises use of a transmembrane delivery system to deliver sdRNA oligonucleotides to a population of PBLs.

[0554] Oligonucleotides and oligonucleotide compositions are contacted with (e.g., brought into contact with, also referred to herein as administered or delivered to) and taken up by PBLs described herein, including through passive uptake by PBLs. The sdRNA can be added to the PBLs as described herein during the step of culturing in the first culture medium, after the step of culturing in the first culture medium, before or during the step of culturing in the second culture medium, before the harvest step, during or after harvest step, before or during the step of final formulation and / or transfer to infusion bag, as well as before any optional cryopreservation step. Moreover, sdRNA can be added after thawing from any cryopreservation step. In an embodiment, one or more sdRNAs targeting genes as described herein, including PD-1, LAG-3, TIM-3, CISH, and CBLB, may be added to cell culture media comprising PBLs and other agents at concentrations selected from the group consisting of 100 nM to 20 mM, 200 nM to 10 mM, 500 nm to 1 mM, 1 μM to 100 μM, and 1 μM to 100 μM. In an embodiment, one or more sdRNAs targeting genes as described herein, including PD-1, LAG-3, TIM-3, CISH, and CBLB, may be added to cell culture media comprising PBLs and other agents at amounts selected from the group consisting of 0.1 μM sdRNA / 10,000 PBLs / 100 μL media, 0.5 μM sdRNA / 10,000 PBLs / 100 μL media, 0.75 μM sdRNA / 10,000 PBLs / 100 μL media, 1 μM sdRNA / 10,000 PBLs / 100 μL media, 1.25 μM sdRNA / 10,000 PBLs / 100 μL media, 1.5 μM sdRNA / 10,000 PBLs / 100 μL media, 2 μM sdRNA / 10,000 PBLs / 100 μL media, 5 μM sdRNA / 10,000 PBLs / 100 μL media, or 10 μM sdRNA / 10,000 PBLs / 100 μL media. In an embodiment, one or more sdRNAs targeting genes as described herein, including PD-1, LAG-3, TIM-3, CISH, and CBLB, may be added to TIL cultures during the culturing steps twice a day, once a day, every two days, every three days, every four days, every five days, every six days, or every seven days.

[0555] Oligonucleotide compositions of the invention, including sdRNA, can be contacted with PBLs as described herein during the expansion process, for example by dissolving sdRNA at high concentrations in cell culture media and allowing sufficient time for passive uptake to occur. In certain embodiments, the method of the present invention comprises contacting a population of PBLs with an oligonucleotide composition as described herein. In certain embodiments, the method comprises dissolving an oligonucleotide e.g. sdRNA in a cell culture media and contacting the cell culture media with a population of PBLs. The PBLs may be a first population, a second population and / or a third population as described herein.

[0556] In some embodiments, delivery of oligonucleotides into cells can be enhanced by suitable art recognized methods including calcium phosphate, DMSO, glycerol or dextran, electroporation, or by transfection, e.g., using cationic, anionic, or neutral lipid compositions or liposomes using methods known in the art (see, e.g., WO 90 / 14074; WO 91 / 16024; WO 91 / 17424; U.S. Pat. No. 4,897,355; Bergan et a 1993. Nucleic Acids Research. 21:3567).

[0557] In some embodiments, more than one sdRNA is used to reduce expression of a target gene. In some embodiments, one or more of PD-1, TIM-3, CBLB, LAG3 and / or CISH targeting sdRNAs are used together. In some embodiments, a PD-1 sdRNA is used with one or more of TIM-3, CBLB, LAG3 and / or CISH in order to reduce expression of more than one gene target. In some embodiments, a LAG3 sdRNA is used in combination with a CISH targeting sdRNA to reduce gene expression of both targets. In some embodiments, the sdRNAs targeting one or more of PD-1, TIM-3, CBLB, LAG3 and / or CISH herein are commercially available from Advirna LLC, Worcester, MA, USA.

[0558] In some embodiments, the sdRNA targets a gene selected from the group consisting of PD-1, LAG3, TIM3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBLB, BAFF (BR3), and combinations thereof. In some embodiments, the sdRNA targets a gene selected from the group consist...

Claims

1. A method of treating a hematological malignancy in a patient, the method comprising administering, to a patient, a therapeutically effective population of peripheral blood lymphocytes (PBLs) obtained from the patient.

2. The method of claim 1, wherein the patient has been pre-treated with an ITK inhibitor, a BTK inhibitor and / or a BCL2 inhibitor to the patient.

3. The method of claim 2, wherein the ITK inhibitor is an ITK inhibitor that covalently binds to ITK.

4. The method of claim 2 or 3, wherein the BTK inhibitor is optionally a BTK inhibitor that covalently binds to BTK.

5. The method of any of claims 2-4, wherein the BCL2 inhibitor is optionally a BCL2 inhibitor that mimics BH3.

6. The method of any of claims 1-5, wherein the patient is suffering from a leukemia or a chronic lymphocytic leukemia.

7. The method of claim 6, wherein the patient has relapsed after treatment with a BTK inhibitor, a BCL2 inhibitor or a combination of a BTK inhibitor and BCL2 inhibitor.

8. The method of claim 6 or 7, wherein the patient is ineligible for CAR-T therapy or has a 17p deletion.

9. The method of any of claims 1-8, wherein the patient is suffering from small lymphocytic lymphoma.

10. The method of claim 9, wherein the patient has relapsed after treatment with a BTK inhibitor, a BCL2 inhibitor or a combination of a BTK inhibitor and BCL2 inhibitor.

11. The method of claim 9 or 10, wherein the patient is ineligible for CAR-T therapy or has a 17p deletion.

12. The method of any of claims 1-11, wherein the hematological malignancy selected from the group consisting of acute myeloid leukemia (AML), AML without genetic markers or without molecular targets, myelodysplastic syndrome (MDS), MDS without genetic markers or without molecular targets, low grade MDS, hypoplastic MDS, EBV related lymphoproliferative disorder, post-transplant lymphoproliferative disorder, CD123 negative (IL-3R negative) AML, FLT3 mutation negative AML, FLT3 positive AML, Nucleophosmin (NPM1) mutation negative acute myeloid leukemia, and NPM1 positive AML.

13. The method of any of claims 1-11, wherein the hematological malignancy selected from the group consisting of the mantle cell lymphoma (MCL), follicular lymphoma (FL), diffuse large B cell lymphoma (DLBCL), activated B cell (ABC) DLBCL, and germinal center B cell (GCB) and DLBCL.

14. The method of any of claims 1-11, wherein the hematological malignancy selected from the group consisting of chronic lymphocytic leukemia (CLL), CLL with Richter's transformation (or Richter's syndrome), CLL replased after treatment with BTK inhibitors, CLL relapsed after treatment with BCL2 inhibitors, CLL replased after treatment with BTK and BCL2 inhibitors, Relapsed CLL with 17p deletion, small lymphocytic leukemia (SLL), SLL replased after treatment with BTK inhibitors, SLL relapsed after treatment with BCL2 inhibitors, SLL replased after treatment with BTK and BCL2 inhibitors, and Relapsed SLL with 17p deletion.

15. The method of any of claims 1-11, wherein the hematological malignancy selected from the group consisting of CD19 negative B cell leukemia or lymphoma (including CLL / SLL), CD20 negative B cell leukemia or lymphoma (including CLL / SLL), CD23 negative B cell leukemia or lymphoma (including CLL / SLL), and CD123 negative B cell leukemia or lymphoma (including CLL / SLL).

16. The method of any of claims 1-11, wherein the hematological malignancy selected from the group consisting of non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, relapsed and / or refractory Hodgkin's lymphoma, B cell acute lymphoblastic leukemia (B-ALL), mature B-ALL, Burkitt's lymphoma, Waldenström's macroglobulinemia (WM), multiple myeloma, myelodysplastic syndromes, myelofibrosis, chronic myelocytic leukemia, follicle center lymphoma, indolent NHL, human immunodeficiency virus (HIV) associated B cell lymphoma, and Epstein-Barr virus (EBV) associated B cell lymphomaEpstein-Barr virus (EBV) associated B cell lymphoma, EBV related lymphoproliferative disorders and post-transplant lymphoproliferative disorders.

17. The method of any of claims 1-16, wherein the patient has been pretreated with a kinase inhibitor selected from the group consisting of imatinib, dasatinib, ibrutinib, bosutinib, nilotinib, erlotinib, acalabrutinib, zanubrutinib, tirabrutinib, orelabrutinib, evobrutinib, spebrutinib, remibrutinib, tolebrutinib, olmutinib, branebrutinib, TAK-020, elsubrutinib, rilzabrutinib, vecabrutinib, fenebrutinib, GNE-431, RN-486, BMS935177, BMS-986142, CGI-1746, GDC-0834, G-744, G-278, and a combination thereof, or a BCL2 inhibitor selected from the group consisting of venetoclax, obatoclax, subatoclax, maritoclax, gossypol, apogossypol, TW-37, UMI-77, BDA-366, navitoclax, ABT-737, and a combinationt hereof.

18. The method of any of claims 1-17, wherein the patient has replapsed after treatment with a kinase inhibitor selected from the group consisting of imatinib, dasatinib, ibrutinib, bosutinib, nilotinib, erlotinib, acalabrutinib, zanubrutinib, tirabrutinib, orelabrutinib, evobrutinib, spebrutinib, remibrutinib, tolebrutinib, olmutinib, branebrutinib, TAK-020, elsubrutinib, rilzabrutinib, vecabrutinib, fenebrutinib, GNE-431, RN-486, BMS935177, BMS-986142, CGI-1746, GDC-0834, G-744, G-278, and a combination thereof, or a BCL2 inhibitor selected from the group consisting of venetoclax, obatoclax, subatoclax, maritoclax, gossypol, apogossypol, TW-37, UMI-77, BDA-366, navitoclax, ABT-737, and a combinationt hereof.

19. The method of any of claims 1-18, wherein the therapeutically effective population of PBLs comprises from about 2.3×1010 to about 13.7×1010 PBLs.

20. A method of treating a hematological malignancy in a patient, the method comprising administering a therapeutically effective population of peripheral blood lymphocytes (PBLs) obtained by the steps comprising:a. obtaining a sample of peripheral blood mononuclear cells (PBMCs) from the peripheral blood of a patient suffering from the hematological malignancy, wherein said sample is optionally cryopreserved and the patient is optionally pretreated with an ITK inhibitor, a BTK inhibitor and / or a BCL2 inhibitor;b. optionally washing the PBMCs by centrifugation,c. adding magnetic beads selective for CD3 and CD28 to the PBMCs;d. seeding PBMCs into a gas-permeable container and co-culturing said PBMCs in media comprising about 3000 IU / mL of IL-2 in for about 4 to about 6 days;e. feeding said PBMCs using media comprising about 3000 IU / mL of IL-2, and co-culturing said PBMCs for about 5 days, such that the total co-culture period of steps d and e is about 9 to about 11 days;f. harvesting PBMCs from media;g. removing the magnetic beads selective for CD3 and CD28 using a magnet;h. removing residual B-cells using magnetic-activated cell sorting and CD19+ beads to provide a peripheral blood lymphocyte (PBL) product;i. washing and concentrating the PBL product using a cell harvester;j. formulating and optionally cryopreserving the PBL product; andk. administering a therapeutically effective dose of the PBL product to the patient,wherein the ITK inhibitor is optionally an ITK inhibitor that covalently binds to ITK,wherein the BTK inhibitor is optionally a BTK inhibitor that covalently binds to BTK, andwherein the BCL2 inhibitor is optionally a BCL2 inhibitor that mimics BH3.

21. The method of claim 20, wherein less than or equal to about 50 mL of peripheral blood of a patient is obtained in step a.

22. The method of claim 20 or 21, wherein between about 10 mL and about 50 mL of peripheral blood of a patient is obtained in step a.

23. The method of any of claims 20-22, wherein the seeding density of PBMCs during step d is about 2×105 / cm2 to about 1.6×103 / cm2 relative to the surface area of the gas-permeable container.

24. A method for treating a hematological malignancy, the method comprising the steps of:(a) obtaining peripheral blood mononuclear cells (PBMCs) from less than or equal to about 50 mL of whole blood from a patient suffering from the hematological malignancy, wherein the patient is optionally pretreated with an ITK inhibitor, a BTK inhibitor and / or a BCL2 inhibitor;(b) admixing beads selective for CD3 and CD28 with the PBMCs, wherein the beads are added at a ratio of 3 beads:1 cell, to form an admixture of PBMCs and beads;(c) culturing the admixture of PBMCs and beads at a density of about 25,000 cells per cm2 to about 50,000 cells per cm2 on a gas-permeable surface of one or more containers containing a first cell culture medium and IL-2 for a period of about 4 days;(d) adding to each container of step (c) IL-2 and a second cell culture medium that is the same as or different from the first cell culture medium and culturing for a period of about 5 days to about 7 days to form an expanded population of peripheral blood lymphocytes (PBLs); and(e) harvesting from each container the expanded population of PBLs; and(f) administering a therapeutically effective portion of the expanded population of PBLs to the patient.

25. The method of claim 24, wherein in step (e) the total number of cells harvested is from about 8 billion to about 22 billion.

26. The method of claim 24 or 25, wherein in step (b) the admixture of beads and PBMCs forms complexes of PBMCs and beads, wherein before step (c) the process comprises the step of separating the complexes from the admixture, and wherein step (c) is replaced by the step of culturing the complexes of PBMCs and beads at a density of about 25,000 cells per cm2 to about 50,000 cells per cm2 on a gas-permeable surface in one or more containers containing a first cell culture medium and IL-2 for a period of about 4 days.

27. The method of claim 26, wherein in step (b) magnetic beads selective for CD3 and CD28 are admixed to the PBMCs, and wherein the step of separating the complexes from the admixture is performed by using a magnet to remove the complexes from the admixture.

28. The method of any of claims 24-27, wherein the beads selective for CD3 and CD28 are beads conjugated to anti-CD3 antibodies and anti-CD28 antibodies.

29. The method of any of claims 24-28, wherein after step (d) the process comprises the step of:(da) performing a selection to remove any remnant B-cells from the expanded population of PBLs.

30. The method of claim 29, wherein in step (da) the selection is performed by using beads selective for CD19 to remove the remnant B-cells.

31. The method of claim 29 or 30, wherein in step (da) the selection is performed by admixing the beads selective for CD19 with the expanded population of PBLs to form complexes of beads and any remnant B-cells and removing the complexes from the admixture.

32. The method of any of claims 29-31, wherein in step (da) the selection is performed by admixing magnetic beads selective for CD19 with the expanded population of PBLs to form complexes of magnetic beads and any remnant B-cells and using a magnet to remove the complexes from the admixture.

33. The method of any of claims 26-32, wherein the beads selective for CD19 are beads conjugated to anti-CD19 antibody.

34. The method of any of claims 24-33, wherein before step (b) the process further comprises the step of removing B-cells from the PBMCs to provide PBMCs depleted of B-cells.

35. The method of any of claims 24-34, wherein before step (b) the process further comprises the step of removing B-cells from the PBMCs by selecting against CD19 to provide PBMCs depleted of B-cells.

36. The method of any of claims 24-35, wherein before step (b) the process further comprises the step of removing B-cells from the PBMCs by admixing beads selective for CD19 with the PBMCs to form complexes of the beads and CD19+ cells in an admixture and removing the complexes from the admixture to provide PBMCs depleted of B-cells.

37. The method of any of claims 24-36, wherein before step (b) the process further comprises the step of removing B-cells from the PBMCs by admixing magnetic beads selective for CD19 with the PBMCs to form complexes of the magnetic beads and CD19+ cells in an admixture and using a magnet to remove the complexes from the admixture to provide PBMCs depleted of B-cells.

38. The method of any of claims 24-37, wherein the first cell culture medium contains about 3000 IU / mL of IL-2.

39. The method of any of claims 24-38, wherein the second cell culture medium contains about 3000 IU / mL of IL-2.

40. The method of any of claims 24-39, wherein in steps (c) and (d) the culture is incubated at 37° C. and under an atmosphere containing 5% CO2.

41. The method of any of claims 24-40, wherein the process is performed over about 9 days.

42. The method of any of claims 24-40, wherein the process is performed over about 11 days.

43. The method of any of claims 24-42, wherein the patient is pretreated with an ITK inhibitor, a BTK inhibitor and / or a BCL2 inhibitor.

44. The method of any of claims 24-43, wherein the patient is pretreated with a kinase inhibitor selected from the group consisting of imatinib, dasatinib, ibrutinib, bosutinib, nilotinib, erlotinib, acalabrutinib, zanubrutinib, tirabrutinib, orelabrutinib, evobrutinib, spebrutinib, remibrutinib, tolebrutinib, olmutinib, branebrutinib, TAK-020, elsubrutinib, rilzabrutinib, vecabrutinib, fenebrutinib, GNE-431, RN-486, BMS935177, BMS-986142, CGI-1746, GDC-0834, G-744, G-278, and a combination thereof, or a BCL2 inhibitor selected from the group consisting of venetoclax, obatoclax, subatoclax, maritoclax, gossypol, apogossypol, TW-37, UMI-77, BDA-366, navitoclax, ABT-737, and a combinationt thereof.

45. The method of any of claims 24-44, wherein the patient is suffering from a leukemia or a chronic lymphocytic leukemia.

46. The method of any of claims 20-23, wherein the patient is suffering from a leukemia or a chronic lymphocytic leukemia.

47. The method of any of claims 20-46, wherein the patient has relapsed after treatment with a BTK inhibitor, a BCL2 inhibitor or a combination of a BTK inhibitor and BCL2 inhibitor.

48. The method of any of claims 20-47, wherein the patient is ineligible for CAR-T therapy or a 17p deletion.

49. The method of any of claims 20-48, wherein the patient has relapsed after treatment with a BTK inhibitor, a BCL2 inhibitor or a combination of a BTK inhibitor and BCL2 inhibitor.

50. The method of any of claims 20-49, wherein the patient is ineligible for CAR-T therapy or a 17p deletion.

51. The method of any of claims 20-50, wherein the patient is suffering from small lymphocytic lymphoma.

52. The method of claim 51, wherein the patient has relapsed after treatment with a BTK inhibitor, a BCL2 inhibitor or a combination of a BTK inhibitor and BCL2 inhibitor.

53. The method of claim 51 or 52, wherein the patient is ineligible for CAR-T therapy or a 17p deletion.

54. The method of any one of claims 20-53, wherein the patient is suffering from the hematological malignancy selected from the group consisting of acute myeloid leukemia (AML), AML without genetic markers or without molecular targets, MDS without genetic markers or without molecular targets, low grade MDS, hypoplastic MDS, CD123 negative (IL-3R negative) AML, FLT3 mutation negative AML, FLT3 positive AML, Nucleophosmin (NPM1) mutation negative acute myeloid leukemia, NPM1 positive AML, mantle cell lymphoma (MCL), follicular lymphoma (FL), diffuse large B cell lymphoma (DLBCL), activated B cell (ABC) DLBCL, germinal center B cell (GCB) DLBCL, chronic lymphocytic leukemia (CLL), CLL with Richter's transformation (or Richter's syndrome), CLL replased after treatment with BTK inhibitors, CLL relapsed after treatment with BCL2 inhibitors, CLL replased after treatment with BTK and BCL2 inhibitors, Relapsed CLL with 17p deletion, small lymphocytic leukemia (SLL), SLL replased after treatment with BTK inhibitors, SLL relapsed after treatment with BCL2 inhibitors, SLL replased after treatment with BTK and BCL2 inhibitors, Relapsed SLL with 17p deletion, CD19 negative B cell leukemia or lymphoma (including CLL / SLL), CD20 negative B cell leukemia or lymphoma (including CLL / SLL), CD23 negative B cell leukemia or lymphoma (including CLL / SLL), non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, relapsed and / or refractory Hodgkin's lymphoma, B cell acute lymphoblastic leukemia (B-ALL), mature B-ALL, Burkitt's lymphoma, Waldenström's macroglobulinemia (WM), multiple myeloma, myelodysplastic syndromes, myelofibrosis, chronic myelocytic leukemia, follicle center lymphoma, indolent NHL, human immunodeficiency virus (HIV) associated B cell lymphoma, and Epstein-Barr virus (EBV) associated B cell lymphomaEpstein-Barr virus (EBV) associated B cell lymphoma, EBV related lymphoproliferative disorders and post-transplant lymphoproliferative disorders.

55. A method of treating a subject with a hematological malignancy, the method comprising administering a peripheral blood lymphocyte (PBL) product obtained by steps comprising:(a) performing a first expansion by culturing an admixture of peripheral blood mononuclear cells (PBMCs) and beads selective for CD3 and CD28 at a density of about 25,000 cells per cm2 to about 50,000 cells per cm2 on a gas-permeable surface of one or more containers containing a first cell culture medium and IL-2 for a period of about 4 days, wherein the PBMCs are obtained from peripheral blood of the subject, wherein the subject is pretreated with an ITK inhibitor, a BTK inhibitor, and / or a BCL2 inhibitor;(b) adding to each container of step (c) IL-2 and a second cell culture medium that is the same as or different from the first cell culture medium and culturing for a period of about 5 days to about 7 days to form an expanded population of peripheral blood lymphocytes (PBLs);(c) harvesting the expanded population of PBLs from each container; and(d) administering a therapeutically effective portion of the expanded population of PBLs to the subject.

56. The method of claim 55, wherein in step (c) the total number of cells harvested is from about 8 billion to about 22 billion.

57. The method of claim 55 or 56, wherein the beads selective for CD3 and CD28 are beads conjugated to anti-CD3 antibodies and anti-CD28 antibodies.

58. The method of any of claims 55-57, wherein after step (b) the process comprises the step of:(ba) performing a selection to remove any remnant B-cells from the expanded population of PBLs.

59. The method of claim 58, wherein in step (ba) the selection is performed by using beads selective for CD19 to remove the remnant B-cells.

60. The method of claim 58 or 59, wherein in step (ba) the selection is performed by admixing the beads selective for CD19 with the expanded population of PBLs to form complexes of beads and any remnant B-cells and removing the complexes from the admixture.

61. The method of any of claims 58-60, wherein in step (ba) the selection is performed by admixing magnetic beads selective for CD19 with the expanded population of PBLs to form complexes of magnetic beads and any remnant B-cells and using a magnet to remove the complexes from the admixture.

62. The method of any of claims 58-61, wherein the beads selective for CD19 are beads conjugated to anti-CD19 antibody.

63. The method of any of claims 55-62, wherein before step (a) the process further comprises the step of removing B-cells from the PBMCs to provide PBMCs depleted of B-cells.

64. The method of any of claims 55-63, wherein before step (a) the process further comprises the step of removing B-cells from the PBMCs by selecting against CD19 to provide PBMCs depleted of B-cells.

65. The method of any of claims 55-64, wherein before step (a) the process further comprises the step of removing B-cells from the PBMCs by admixing beads selective for CD19 with the PBMCs to form complexes of the beads and CD19+ cells in an admixture and removing the complexes from the admixture to provide PBMCs depleted of B-cells.

66. The method of any of claims 55-65, wherein before step (a) the process further comprises the step of removing B-cells from the PBMCs by admixing magnetic beads selective for CD19 with the PBMCs to form complexes of the magnetic beads and CD19+ cells in an admixture and using a magnet to remove the complexes from the admixture to provide PBMCs depleted of B-cells.

67. The method of any of claims 55-66, wherein the first cell culture medium contains about 3000 IU / mL of IL-2.

68. The method of any of claims 55-67, wherein the second cell culture medium contains about 3000 IU / mL of IL-2.

69. The method of any of claims 55-68, wherein in steps (a) and (b) the culture is incubated at 37° C. and under an atmosphere containing 5% CO2.

70. The method of any of claims 55-69, wherein the process is performed over about 9 days.

71. The method of any of claims 55-69, wherein the process is performed over about 11 days.

72. The method of any of claims 55-71, wherein the patient is pretreated with ibrutinib.

73. The method of any of claims 55-72, wherein the patient is suffering from a leukemia or a chronic lymphocytic leukemia.

74. The method of any of claims 55-73, wherein the patient has relapsed after treatment with a BTK inhibitor, a BCL2 inhibitor or a combination of a BTK inhibitor and BCL2 inhibitor.

75. The method of any of claims 55-74, wherein the patient is ineligible for CAR-T therapy or a 17p deletion.

76. The method of any one of claims 55-75, wherein the patient is suffering from the hematological malignancy selected from the group consisting of acute myeloid leukemia (AML), AML without genetic markers or without molecular targets, MDS without genetic markers or without molecular targets, low grade MDS, hypoplastic MDS, CD123 negative (IL-3R negative) AML, FLT3 mutation negative AML, FLT3 positive AML, Nucleophosmin (NPM1) mutation negative acute myeloid leukemia, NPM1 positive AML, mantle cell lymphoma (MCL), follicular lymphoma (FL), diffuse large B cell lymphoma (DLBCL), activated B cell (ABC) DLBCL, germinal center B cell (GCB) DLBCL, chronic lymphocytic leukemia (CLL), CLL with Richter's transformation (or Richter's syndrome), CLL replased after treatment with BTK inhibitors, CLL relapsed after treatment with BCL2 inhibitors, CLL replased after treatment with BTK and BCL2 inhibitors, Relapsed CLL with 17p deletion, small lymphocytic leukemia (SLL), SLL replased after treatment with BTK inhibitors, SLL relapsed after treatment with BCL2 inhibitors, SLL replased after treatment with BTK and BCL2 inhibitors, Relapsed SLL with 17p deletion, CD19 negative B cell leukemia or lymphoma (including CLL / SLL), CD20 negative B cell leukemia or lymphoma (including CLL / SLL), CD23 negative B cell leukemia or lymphoma (including CLL / SLL), non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, relapsed and / or refractory Hodgkin's lymphoma, B cell acute lymphoblastic leukemia (B-ALL), mature B-ALL, Burkitt's lymphoma, Waldenström's macroglobulinemia (WM), multiple myeloma, myelodysplastic syndromes, myelofibrosis, chronic myelocytic leukemia, follicle center lymphoma, indolent NHL, human immunodeficiency virus (HIV) associated B cell lymphoma, and Epstein-Barr virus (EBV) associated B cell lymphomaEpstein-Barr virus (EBV) associated B cell lymphoma, EBV related lymphoproliferative disorders and post-transplant lymphoproliferative disorders.

77. A method of treating a subject with a hematological malignancy, the method comprising administering a peripheral blood lymphocyte (PBL) product comprising:(a) obtaining an admixture of peripheral blood mononuclear cells (PBMCs) and beads selective for CD3 and CD28, wherein the PBMCs are obtained from peripheral blood of the subject, wherein the subject is pretreated with an ITK inhibitor, a BTK inhibitor, and / or a BCL2 inhibitor;(b) separating complexes PBMCs and beads from the admixture;(c) culturing the complexes of PBMCs and beads at a density of about 25,000 cells per cm2 to about 50,000 cells per cm2 on a gas-permeable surface in one or more containers containing a first cell culture medium and IL-2 for a period of about 4 days;(d) adding to each container of step (c) IL-2 and a second cell culture medium that is the same as or different from the first cell culture medium and culturing for a period of about 5 days to about 7 days to form an expanded population of peripheral blood lymphocytes (PBLs);(e) harvesting the expanded population of PBLs from each container; and(f) administering a therapeutically effective portion of the expanded population of PBLs to the subject.

78. The method of claim 77, wherein in step (a) magnetic beads selective for CD3 and CD28 are admixed to the PBMCs, and wherein the step (b) is performed by using a magnet to remove the complexes from the admixture.

79. The method of claim 77 or 78, wherein in step (e) the total number of cells harvested is from about 8 billion to about 22 billion.

80. The method of any of claims 77-79, wherein the beads selective for CD3 and CD28 are beads conjugated to anti-CD3 antibodies and anti-CD28 antibodies.

81. The method of any of claims 77-80, wherein after step (a) the process comprises the step of:(aa) performing a selection to remove any remnant B-cells from the expanded population of PBLs.

82. The method of claim 81, wherein in step (aa) the selection is performed by using beads selective for CD19 to remove the remnant B-cells.

83. The method of claim 81, wherein in step (aa) the selection is performed by admixing the beads selective for CD19 with the expanded population of PBLs to form complexes of beads and any remnant B-cells and removing the complexes from the admixture.

84. The method of claim 83, wherein in step (aa) the selection is performed by admixing magnetic beads selective for CD19 with the expanded population of PBLs to form complexes of magnetic beads and any remnant B-cells and using a magnet to remove the complexes from the admixture.

85. The method of claim 83, wherein the beads selective for CD19 are beads conjugated to anti-CD19 antibody.

86. The method of any of claims 77-85, wherein before step (a) the process further comprises the step of removing B-cells from the PBMCs to provide PBMCs depleted of B-cells.

87. The method of any of claims 77-86, wherein before step (a) the process further comprises the step of removing B-cells from the PBMCs by selecting against CD19 to provide PBMCs depleted of B-cells.

88. The method of any of claims 77-87, wherein before step (a) the process further comprises the step of removing B-cells from the PBMCs by admixing beads selective for CD19 with the PBMCs to form complexes of the beads and CD19+ cells in an admixture and removing the complexes from the admixture to provide PBMCs depleted of B-cells.

89. The method of any of claims 77-88, wherein before step (a) the process further comprises the step of removing B-cells from the PBMCs by admixing magnetic beads selective for CD19 with the PBMCs to form complexes of the magnetic beads and CD19+ cells in an admixture and using a magnet to remove the complexes from the admixture to provide PBMCs depleted of B-cells.

90. The method of any of claims 77-89, wherein the first cell culture medium contains about 3000 IU / mL of IL-2.

91. The method of any of claims 77-90, wherein the second cell culture medium contains about 3000 IU / mL of IL-2.

92. The method of any of claims 77-91, wherein in steps (a) and (b) the culture is incubated at 37° C. and under an atmosphere containing 5% CO2.

93. The method of any of claims 77-92, wherein the process is performed over about 9 days.

94. The method of any of claims 77-92, wherein the process is performed over about 11 days.

95. The method of any of claims 77-94, wherein the patient is pretreated with a kinase inhibitor selected from the group consisting of imatinib, dasatinib, ibrutinib, bosutinib, nilotinib, erlotinib, acalabrutinib, zanubrutinib, tirabrutinib, orelabrutinib, evobrutinib, spebrutinib, remibrutinib, tolebrutinib, olmutinib, branebrutinib, TAK-020, elsubrutinib, rilzabrutinib, vecabrutinib, fenebrutinib, GNE-431, RN-486, BMS935177, BMS-986142, CGI-1746, GDC-0834, G-744, G-278, and a combination thereof, or a BCL2 inhibitor selected from the group consisting of venetoclax, obatoclax, subatoclax, maritoclax, gossypol, apogossypol, TW-37, UMI-77, BDA-366, navitoclax, ABT-737, and a combinationt hereof.

96. The method of any of claims 77-95, wherein the patient is suffering from a leukemia or a chronic lymphocytic leukemia.

97. The method of any of claims 77-96, wherein the patient has relapsed after treatment with a BTK inhibitor, a BCL2 inhibitor or a combination of a BTK inhibitor and BCL2 inhibitor.

98. The method of any of claims 77-96, wherein the patient is ineligible for CAR-T therapy or a 17p deletion.

99. The method of any of claims 77-98, wherein the patient is suffering from the hematological malignancy selected from the group consisting of acute myeloid leukemia (AML), AML without genetic markers or without molecular targets, MDS without genetic markers or without molecular targets, low grade MDS, hypoplastic MDS, CD123 negative (IL-3R negative) AML, FLT3 mutation negative AML, FLT3 positive AML, Nucleophosmin (NPM1) mutation negative acute myeloid leukemia, NPM1 positive AML, mantle cell lymphoma (MCL), follicular lymphoma (FL), diffuse large B cell lymphoma (DLBCL), activated B cell (ABC) DLBCL, germinal center B cell (GCB) DLBCL, chronic lymphocytic leukemia (CLL), CLL with Richter's transformation (or Richter's syndrome), CLL replased after treatment with BTK inhibitors, CLL relapsed after treatment with BCL2 inhibitors, CLL replased after treatment with BTK and BCL2 inhibitors, Relapsed CLL with 17p deletion, small lymphocytic leukemia (SLL), SLL replased after treatment with BTK inhibitors, SLL relapsed after treatment with BCL2 inhibitors, SLL replased after treatment with BTK and BCL2 inhibitors, Relapsed SLL with 17p deletion, CD19 negative B cell leukemia or lymphoma (including CLL / SLL), CD20 negative B cell leukemia or lymphoma (including CLL / SLL), CD23 negative B cell leukemia or lymphoma (including CLL / SLL), non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, relapsed and / or refractory Hodgkin's lymphoma, B cell acute lymphoblastic leukemia (B-ALL), mature B-ALL, Burkitt's lymphoma, Waldenström's macroglobulinemia (WM), multiple myeloma, myelodysplastic syndromes, myelofibrosis, chronic myelocytic leukemia, follicle center lymphoma, indolent NHL, human immunodeficiency virus (HIV) associated B cell lymphoma, and Epstein-Barr virus (EBV) associated B cell lymphomaEpstein-Barr virus (EBV) associated B cell lymphoma, EBV related lymphoproliferative disorders and post-transplant lymphoproliferative disorders.

100. A method of treating a subject with a hematological malignancy, the method comprising administering a peripheral blood lymphocyte (PBL) product comprising:(a) performing a first expansion by seeding PBMCs into a gas-permeable container and co-culturing the PBMCs with magnetic beads selective for CD3 and CD28 in media comprising about 3000 IU / mL of IL-2 in for about 4 to about 6 days, wherein the PBMCs are obtained from peripheral blood of the subject, wherein the subject is pretreated with an ITK inhibitor, a BTK inhibitor, and / or a BCL2 inhibitor;(b) performing a second expansion by feeding the co-culture using media comprising about 3000 IU / mL of IL-2, and co-culturing said PBMCs for about 5 days, such that the total co-culture period of steps (a) and (b) is about 9 to about 11 days;(c) harvesting PBMCs from media;(d) removing the magnetic beads selective for CD3 and CD28 using a magnet;(e) removing residual B-cells using magnetic-activated cell sorting and CD19+ beads to provide a peripheral blood lymphocyte (PBL) product; and(f) administering a therapeutically effective dose of a formulation comprising the PBL product to the subject.

101. The method of claim 100, wherein the ITK inhibitor is optionally an ITK inhibitor that covalently binds to ITK.

102. The method of claim 100 or 101, wherein the BTK inhibitor is optionally a BTK inhibitor that covalently binds to BTK.

103. The method of any of claims 100-102, wherein the BCL2 inhibitor is optionally a BCL2 inhibitor that mimics BH3.

104. The method of any of claims 100-103, wherein the amount of peripheral blood obtained from the patient for step (a) is less than or equal to about 50 mL.

105. The method of any of claims 100-104, wherein the amount of peripheral blood obtained from the patient for step (a) is between about 10 mL and about 50 mL.

106. The method of any of claims 100-105, wherein a seeding density of PBMCs during step (a) is about 2×105 / cm2 to about 1.6×103 / cm2 relative to the surface area of the gas-permeable container.

107. The method of any of claims 100-106, wherein the patient is suffering from a leukemia or a chronic lymphocytic leukemia.

108. The method of any of claims 100-107, wherein the patient is ineligible for CAR-T therapy or a 17p deletion.

109. The method of any of claims 100-108, wherein the patient is suffering from small lymphocitic lymphoma.

110. The method of any of claims 100-109, wherein the patient has relapsed after treatment with a BTK inhibitor, a BCL2 inhibitor or a combination of a BTK inhibitor and BCL2 inhibitor.

111. The method of any of claims 100-110, wherein the patient is suffering from the hematological malignancy selected from the group consisting of acute myeloid leukemia (AML), AML without genetic markers or without molecular targets, MDS without genetic markers or without molecular targets, low grade MDS, hypoplastic MDS, CD123 negative (IL-3R negative) AML, FLT3 mutation negative AML, FLT3 positive AML, Nucleophosmin (NPM1) mutation negative acute myeloid leukemia, NPM1 positive AML, mantle cell lymphoma (MCL), follicular lymphoma (FL), diffuse large B cell lymphoma (DLBCL), activated B cell (ABC) DLBCL, germinal center B cell (GCB) DLBCL, chronic lymphocytic leukemia (CLL), CLL with Richter's transformation (or Richter's syndrome), CLL replased after treatment with BTK inhibitors, CLL relapsed after treatment with BCL2 inhibitors, CLL replased after treatment with BTK and BCL2 inhibitors, Relapsed CLL with 17p deletion, small lymphocytic leukemia (SLL), SLL replased after treatment with BTK inhibitors, SLL relapsed after treatment with BCL2 inhibitors, SLL replased after treatment with BTK and BCL2 inhibitors, Relapsed SLL with 17p deletion, CD19 negative B cell leukemia or lymphoma (including CLL / SLL), CD20 negative B cell leukemia or lymphoma (including CLL / SLL), CD23 negative B cell leukemia or lymphoma (including CLL / SLL), non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, relapsed and / or refractory Hodgkin's lymphoma, B cell acute lymphoblastic leukemia (B-ALL), mature B-ALL, Burkitt's lymphoma, Waldenström's macroglobulinemia (WM), multiple myeloma, myelodysplastic syndromes, myelofibrosis, chronic myelocytic leukemia, follicle center lymphoma, indolent NHL, human immunodeficiency virus (HIV) associated B cell lymphoma, and Epstein-Barr virus (EBV) associated B cell lymphomaEpstein-Barr virus (EBV) associated B cell lymphoma, EBV related lymphoproliferative disorders and post-transplant lymphoproliferative disorders.

112. The method of any one of the preceding claims, wherein prior to administering the therapeutically effective dose, the patient is administered a non-myeloablative lymphodepletion treatment.

113. A use of a therapeutically effective population of peripheral blood lymphocytes (PBLs) for treating a patient with a hematological malignancy, the use comprising administering to the patient the therapeutically effective population of PBLs, wherein the therapeutic population of PBLs is obtained from the patient.

114. The use of claim 113, wherein the patient has been pretreated with an ITK inhibitor, a BTK inhibitor and / or a BCL2 inhibitor.

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